Arabidopsis, three KDEL CysEPs  are expressed. We have previously shown that AtCEP1 is a factor of basal resistance to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum, and is expressed in spatiotemporal association with the late fungal development on Arabidopsis leaves. The endoplasmic reticulum-localized proenzyme of AtCEP1 was further visualized at the haustorial complex encased with callose. The AtCPR5 gene (CONSTITUTIVE EXPRESSION OF PR GENES 5) is a regulator of expression of pathogenesis related genes. Loss of AtCPR5 leads to spontaneous expression of chlorotic lesions which was associated with enhanced expression of AtCEP1. We used the atcpr5-2 mutant plants and the atcep1 atcpr5-2 double mutants harboring a non-functional reporter (PCEP1::pre-pro-3xHA-EGFP-KDEL) for visualization of AtCEP1 promoter activity. We found the specific up-regulation of AtCEP1 in direct neighborhood of spreading leaf lesions thus likely representing cells undergoing PCD. Furthermore, we found a strong resistance of atcpr5 mutant plants against infection with E. cruciferarum. Loss of AtCEP1 had no obvious influence on the strong resistance of atcpr5-2 mutant plants against infection with E. cruciferarum. However, the area of necrotic leaf lesions associated with E. cruciferarum colonies was significantly larger in atcpr5-2 as compared to atcep1 atcpr5-2 double mutant plants. The presence of AtCEP1 thus contributes to AtCPR5-controlled PCD at the sites of powdery mildew infection.Programmed cell death (PCD) is a prerequisite for successful development and it limits the spread of biotrophic pathogens in a rapid hypersensitive response at the site of infection. KDEL-tailed cysteine endopeptidases (KDEL CysEP) are a subgroup of papain-type cysteine endopeptidases expressed in tissues undergoing PCD. In  Programmed cell death (PCD) is a genetically determined, highly regulated process in all multicellular organisms and a prerequisite for successful development. PCD eliminates tissues and cells serving temporary functions during development such as tapetum cells in anthers and suspensor cells connecting the embryo to the mother plant or nucellus cells of a mature ovule –4. PlantRicinus communis) as the founding member [Diverse classes of proteases are involved in PCD, including cysteine proteases, serine proteases, aspartic proteases and metalloproteases ,7. A unig member –10. KDELg member . KDEL Cyg member –14; upong member  (as descg member ). The mag member ). The brg member . The resg member .Arabidopsis, three KDEL CysEPs—CEP1 (At5g50260), CEP2 (At3g48340), and CEP3 (At3g48350)—have been identified that are expressed in tissues undergoing developmental PCD  showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of CEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous CEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) rescued the pathogenesis phenotype demonstrating the function of CEP1 in restriction of powdery mildew disease. atcep1 knockout plants transformed with the non-functional reporter including EGFP without the mature CEP1 subunit (PCEP1::pre-pro-3xHA-EGFP-KDEL) retained susceptibility to E. cruciferarum. The spatiotemporal CEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of CEP1 in controlling late stages of the compatible interaction including late epidermal cell death . Fureviously ). Two CEeviously ).Arabidopsis are regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) function as key signaling molecules. Mutants such as cpr5 constitutively activate these defense pathways [CPR5 gene  is a regulator of expression of pathogenesis related genes and participates in signal transduction pathways involved in plant defense and development such as leaf senescence or flowering. It codes for nuclear envelope membrane protein [CPR5 leads to spontaneous expression of chlorotic lesions and reduced trichome development [cpr5 plants were found to be constitutively resistant to virulent pathogens such as the bacterial pathogen Pseudomonas syringae and the oomycete Hyaloperonospora arabidopsidis [CEP1  is constitutively up-regulated in cpr5 mutants [www.genevestigator.com; cpr5-2 mutant allele that has a point mutation in the fourth exon leading to a premature stop codon (Trp477stop) [CEP1 upregulation to chlorotic leaf lesions in cpr5. We measured an increase in CEP1 expression in cpr5-2 mutant, which coincided with the appearance of leaf lesions. The expression of CEP1 was particularly evidenced in leaf cells that surround the chlorotic lesions and presumably underwent cell death. Furthermore, we found a strong resistance of cpr5-2 against infection with E. cruciferarum and studied the pathogenesis and cell death phenotypes in cep1 cpr5-2 double mutants as compared to the single mutants. This suggests a contribution of CEP1 to CPR5-controlled cell death.Defense responses in pathways . The CPR protein . Loss ofelopment ,22. The dopsidis ,21. We f477stop)  in ordercep1  [CEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL that rescued the cep1 knockout phenotype when expressed in cep1 [We used homozygous knockout mutants for rd exon) . For ima in cep1 .cpr5-2 mutant allele (NASC stock code N3770) with a point mutation in the fourth exon leading to a stop codon (Tpr477stop) [GGCTCCTCGTAAGTGTCTTCAGC and cpr5-2 rv GGTCTGACTATGCTTGAGACGAG. The resulting PCR product was sequenced using the primers cpr5-2 seq fw CGATCATCAGGTACGAAGC and cpr5-2 seq rv GTCACGTTTATAGGACCG. Homozygous cep1 cpr5-2 double mutant plants were obtained by crossing. In order to monitor CEP1 promotor activity in cells of the cpr5-2 mutant background, we made homozygous double mutants by crossing cpr5 mutants and cep1 knockout mutants harboring the non-functional reporter PCEP1::pre-pro-3xHA-EGFP-KDEL that exhibits a translational fusion protein of the necessary CEP1 targeting sequences together with a three-fold hemaglutinin-tag and the green fluorescent protein EGFP but lacking the mature CEP1 subunit under control of the endogenous CEP1 promoter [A homozygous 477stop)  was obtapromoter .Inoculation and assessment of disease progression was carried out as described .Arabidopsis cells were visualized by methyl blue  staining. For this purpose, leaves of cpr5-2 single mutant plants and of cep1 cpr5-2 double mutant plants, respectively, were harvested 5 days post inoculation (dpi) with E. cruciferarum and were discoloured in ethanol-acetic acid glacial . The discoloured leaves were rinsed with distilled water and transferred into 67 mM K2HPO4 buffer for 10 minutes followed by incubation for 2 h in the staining solution (0.05% methyl blue in 67 mM K2HPO4) in the dark and by direct analysis using fluorescence microscopy .Callose depositions in cpr5-2 single mutant plants and on cep1 cpr5-2 double mutant plants, respectively, were photographed and the fluorescing areas were quantified using ImageJ graphical analysis software version 1.48v [cpr5-2 single mutant plants and of cep1 cpr5-2 double mutant plants, respectively, were measured using their fluorescence intensity. These values were subtracted from the fluorescent areas of necrotic leaf lesions associated with powdery mildew colonies on cpr5-2 single mutant plants and on cep1 cpr5-2 double mutant plants, respectively, thus obtaining the area of a chlorotic leaf lesion associated with a powdery mildew colony.Only areas of necrotic leaf lesions associated with powdery mildew colonies were measured using their fluorescence intensity. Areas as measured by fluorescence intensity under excitation by 340 nm wave length thus represented necrotic cells, but also other callose-enriched structures such as encasements of established haustoria. Sixty five fluorescing areas detected on 19 leaves on on 1.48v . To thisTGAGACCTTTAATTCTCCAGCTATG; ACT8 qRT rv: CCAGAGTCCAACACAATACCG; CEP1 qRT fw: TCAGCCTGTTTCTGTTGCTATT; CEP1 qRT rv: CATCTCCCGGTAAACACTCC; CEP2 qRT fw: GCTGTTGCAAACCAACCTG; CEP2 qRT rv: TTCCACAAGATCCCGTAAACA; CEP3 qRTfw: GCTCACCAGCCTGTCTCTGT; CEP3 qRT rv: CGCATTCTCCGATAAACACA. qRT-PCR was carried out as described [Primers used for qRT-PCR are: ACT8 qRT fw: escribed .CEP1::pre-pro-3xHA-EGFP-KDEL) or functional (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) reporter plants was carried out as described [Staining of callose and imaging of the non-functional  and accumulate it around haustoria. This led to the speculation that CEP1 could limit haustorium functions, which could explain enhanced growth of E. cruciferarum on cep1 mutant plants [CEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL reporter plants with imaging of callose after staining with the aniline dye methyl blue at 11 dpi. Three dimensional reconstructions of fluorescence signals recorded by CLSM suggested that the proenzyme of CEP1 is closely associated with callose-encased haustoria and can be even found within encasements of haustorial complexes (We previously described that cells attacked by t plants . To get omplexes . Closer omplexes . The ER Arabidopsis atcpr5 mutant (GEO accession GSE5745) displayed in Genevestigator [CEP1 transcripts are significantly more abundant in two-week-old cpr5-2 mutants than in wild type. CPR5 is considered as a negative control element of plant programmed cell death and effector-triggered immunity through cell cycle control [cpr5-2 mutant allele that has a point mutation in the fourth exon leading to a stop codon (Trp477stop) [CEP1 in the cpr5-2 mutant allele correlating to the appearance of leaf lesions. CEP1 was not expressed to higher values in leaves of seven day old cpr5-2 seedlings before lesions were visible  ,25. Quan visible  left. An lesions  right. Oormation . When wenscripts . Again, cep1 knockout mutants harboring the non-functional reporter PCEP1::pre-pro-3xHA-EGFP-KDEL and cpr5-2 mutants in order to visualize leaf cells that would express CEP1 (cep1 knockout mutation on the cpr5-mediated resistance or cell death phenotype (see below). Strikingly, promoter activity of CEP1 was exclusively confined to cells in direct neighborhood of chlorotic lesions  possiblCPR5 as a possible regulator of CEP1 expression. Our targeted expression analyses of CEP1 in young asymptomatic and older leaves showing spontaneous leaf lesion development supported that CEP1 is overexpressed during de-regulated PCD in cep5 mutants. With the findings that CPR5 controls cell cycle and PCD during development and effector triggered immunity to leaf pathogens of Arabidopsis [E. cruciferarum-induced leaf cell death albeit in the opposite direction. This was associated with near-complete powdery mildew resistance of cpr5-2 mutants, which appears, based on previously published and our data, generally resistant to many hemibiotrophic and biotrophic pathogens [Publicly available gene expression data drew our attention to bidopsis ,27 and tathogens ,27,33,34CPR5 and CEP1 retained powdery mildew resistance and spontaneous lesion phenotype observed in the cpr5-2 single mutant. Hence, the cpr5-2 mutant phenotype was largely epistatic over that of the cep1 phenotype in terms of symptom formation of powdery mildew. However, microscopic inspections of the size of lesions that were associated with powdery mildew microcolony formation showed that CEP1 indeed contributed to lesion formation in cpr5-2 mutants challenged by E. cruciferarum. Because these lesions have never been observed to a comparable extent in Col-0 before when challenged by E. cruciferarum and extended into the mesophyll tissue of atcpr5 mutants, we conclude that CPR5 negatively controls a powdery mildew triggered form of PCD. This type of PCD is simultaneously supported by CEP1, such that the cep1 cpr5-2 double mutant showed limited spreading of pathogen triggered cell death before leaf lesions became visible by naked eye. Together this supports that CEP1 acts in pathogen-triggered and CPR5-controlled leaf cell death although it does not represent a central switch in that type of PCD because visible lesions still appeared to an extent not readily distinguishable from that of the atcpr5 single mutant. This study was carried out in an accession of Arabidopsis susceptible to powdery mildew. Since CPR5 acts in effector triggered immunity [Arabidopsis. Spatial association of CEP1 expression with powdery mildew infection sites or CPR5 controlled cell death reactions might be interesting in context of other genes that function in spatial association with cell death. For instance, the salicylic acid receptors NPR3 and NPR4 support or restrict cell death via the abundance of NPR1 in a manner that depends on salicylic acid gradients in spatial distance to the actual site of lesion formation [Double loss of function mutants of immunity ,27, it wimmunity ,36 in Arormation . It mighE. cruciferarum in close spatial association with the fungal haustorium and haustorial callose encasements. Additionally, CPR5 controls resistance to powdery mildew and PCD in response to infection by E. cruciferarum. In cpr5 mutants, CEP1 is overexpressed in spatiotemporal association with spontaneous cell death. CEP1 contributes to CPR5-controlled PCD triggered by E. cruciferarum but is not required to express high level powdery mildew resistance of cpr5 mutants.Papain-type CysEPs have diverse functions in plant defense to pathogens . Our dat


A receptor subunit levels, composition, and regional and subcellular localization. Rapid down-regulation of early responder δ subunit-containing GABAA receptor subtypes mediating ethanol-sensitive tonic inhibitory currents in critical neuronal circuits corresponds to rapid tolerance to ethanol’s behavioral responses. Slightly slower, α1 subunit-containing GABAA receptor subtypes mediating ethanol-insensitive synaptic inhibition are down-regulated, corresponding to tolerance to additional ethanol behaviors plus cross-tolerance to other GABAergic drugs including benzodiazepines, anesthetics, and neurosteroids, especially sedative-hypnotic effects. Compensatory up-regulation of synaptically localized α4 and α2 subunit-containing GABAA receptor subtypes, mediating ethanol-sensitive synaptic inhibitory currents follow, but exhibit altered physio-pharmacology, seizure susceptibility, hyperexcitability, anxiety, and tolerance to GABAergic positive allosteric modulators, corresponding to heightened alcohol withdrawal syndrome. All these changes  induced by ethanol administration are transient and return to normal in a few days. After chronic intermittent ethanol (CIE) treatment the same changes are observed but they become persistent after 30 or more doses, lasting for at least 120 days in the rat, and probably for life. We conclude that the ethanol-induced changes in GABAA receptors represent aberrant plasticity contributing critically to ethanol dependence and increased voluntary consumption. We suggest that the craving, drug-seeking, and increased consumption in the rat model are tied to ethanol-induced plastic changes in GABAA receptors, importantly the development of ethanol-sensitive synaptic GABAA receptor-mediating inhibitory currents that participate in maintained positive reward actions of ethanol on critical neuronal circuits. These probably disinhibit nerve endings of inhibitory GABAergic neurons on dopamine reward circuit cells, and limbic system circuits mediating anxiolysis in hippocampus and amygdala. We further suggest that the GABAA receptors contributing to alcohol dependence in the rat and presumably in human alcohol use disorders (AUD) are the ethanol-induced up-regulated subtypes containing α4 and most importantly α2 subunits. These mediate critical aspects of the positive reinforcement of ethanol in the dependent chronic user while alleviating heightened withdrawal symptoms experienced whenever ethanol is absent. The speculative conclusions based on firm observations are readily testable.GABAergic inhibitory transmission is involved in the acute and chronic effects of ethanol on the brain and behavior. One-dose ethanol exposure induces transient plastic changes in GABA Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence clinically defined as drinking—or being sick from drinking—that interferes with taking care of one’s home or family, or causes job troubles, or school problems, creating large problems both for society and for the drinkers themselves , 2. AUD A receptors (GABAARs) have long been implicated in mediating at least part of the actions of ethanol (EtOH) in mammalian brain. The molecular mechanism(s) of action for intoxicating doses of EtOH have been especially of interest even before the advent of the Research Society on Alcoholism. In recent years, however, the focus of EtOH research has shifted to identifying a protein receptor-based target for EtOH, and several ligand-gated ion channels (LGIC), which include NMDA- Ro15–4513 in forebrain and cerebellum Ro15–4513 to diazepam-insensitive sites in cerebellum and forebrain, considered to involve the α6 and α4 subunits, respectively; we also showed GABAAR subunit mRNA level changes consistent with altered expression [We showed that in CIE, GABAhibition  that washibition  presentethalamus . This isncreased , 129. Inpression .AR subunit composition and function, playing a crucial role in EtOH withdrawal symptoms and dependence. We showed [AR-mediated extrasynaptic current  assays, we found that CIE led to elevated mRNA levels for γ2S but not γ2L, as well as γ1 subunit but not α2; CIE-treated animals were shown to exhibit increased anxiety in the elevated plus maze assay, and behavioral tolerance to the sedative action of EtOH, BZ, and neurosteroids [AR synaptic and tonic inhibitory currents in hippocampal neurons recorded by patch-clamp electrodes in slices from CIE rats [Using subunit-specific antibodies, we measured GABAsteroids . SteroidCIE rats .The changes found after CIE treatment did not appear to involve any gross pathology in either brain or liver . MicroscAR-PAMs: Smith [AR plastic changes induced by EtOH [CIE rats exhibit impaired hippocampal-specific spatial learning deficits , possibls: Smith ) may be s: Smith , 137, an by EtOH , 138. CIAR plasticity induced by CIE, we attempted to learn molecular mechanisms and functional relevance through studies to determine the minimum dose, duration, and frequency of EtOH administration required to produce the changes. We found that a single high, intoxicating, dose of EtOH administered by gavage was able to induce many of the same changes in behavior, GABAAR subunit composition, and hippocampal neuron pharmacology seen in CIE, but the changes were transient [ARs [With the observations of remarkable GABAransient . Thus, went [ARs  are the ent [ARs . All theAR plasticity induced by CIE demonstrated a correlation between the degree of tolerance induced for a series of GABAergic sedative-hypnotic drugs to produce LORR and the degree of tolerance induced for the same drugs to enhance GABAAR-mediated tonic inhibitory currents in hippocampal neurons [AR PAMs .AR-mediated mIPSCs to modulation by low mM EtOH in the recording chamber [ARs, including movement of the α4 into the postsynaptic membrane. The δ subunit was not elevated and did not accumulate in the synaptic membrane, and the increased EtOH modulation of mIPSCs was also observed in the alcohol- naïve knockout (KO) mouse for both GABAAR α4 subunit [ARs are apparently up-regulated by EtOH treatment, and might be positioned in brain locations where they might mediate the continued EtOH-sensitivity in regions needed for the positive reinforcement reward as well as anxiolytic efficacy of EtOH in the dependent individual, animal or human. Therefore, we asked: what might be the subunit composition of GABAARs accounting for this increased sensitivity to EtOH of mIPSCs? Below we describe our discovery of a GABAAR subtype that meets these requirements.However, the CIE rodents and the one-dose EtOH-treated animals show elevated sensitivity of GABA chamber . In CIE  subunit  and δ su subunit –143. FurAR plasticity as in rats. The goal is to establish a short term intermittent EtOH (SIE) mouse model to replicate the information found in the established chronic intermittent EtOH (CIE) model. In comparison to the CIE model, the SIE mouse model can be more easily combined with genetic technology for in depth studies of the underlying mechanisms of alcoholism. C57Bl/6 mice were separated into short intermittent vehicle (SIV) and SIE groups. SIV and SIE mice were gavaged drinking water or ethanol respectively, every other day for five doses and from day 11th, once a day for 30-d. SIV mice served as the control group. We evaluated behavioral changes after two day and 40-d withdrawal from SIE and compared with CIE. The results are consistent with previous reports and indicate that SIE mice, like CIE rats, have greater anxiety, hyperexcitability, and tolerance to acute EtOH-induced LORR than SIV [AR subunits knocked out, in, or down [AR-mediated tonic inhibition throughout the brain and reduced electrophysiological and behavioral effects of gaboxadol [blunted effect of CIE treatment, especially elimination of tolerance development to the intoxicating effects of EtOH, including sedative-hypnotic, motor-incoordinating effects [We have also extended the CIE model to the mouse ; the mouthan SIV . Then we or down , 145. Thaboxadol , includiaboxadol , but, diaboxadol , 142. We effects . We exte effects , where c effects .ARs by EtOH exposure involves protein internalization (endocytosis). In the case of δ subunit, this is clathrin-dependent [AR as early responders to low millimolar EtOH, and likely requires a conformational change in the intracellular domain of δ to allow it to bind the clathrin accessory subunit when the GABAAR protein binds GABA ‘too long,’ as when the GABA concentration is prolonged at a high concentration, or by the presence of a PAM like EtOH to enhance GABA binding. Terunuma et al. [AR molecules exhibit internalization triggered by the prolonged activated protein conformation with the β3 subunit becoming a substrate for a phosphatase that removes phosphate and allowing endocytosis. This mechanism was ruled out for the δ-containing GABAAR [ARs reverts to normal after some hours to days of EtOH removal but fails to normalize after multi-dose CIE regimen [AR δ subunit without change in total δ protein [Both covalent biotinylation of cell surface proteins (technique most suitable for cultured monodisperse cells) and crosslinking of cell surface proteins, to exclude them from the SDS gel during electrophoresis (technique most suitable for brain slices) that the rapid, within hours, and likely minutes, down-regulation of α4βδ GABAependent . This isa et al.  showed tg GABAAR . The dow regimen , 81. We  protein .AR pharmacology and expression related to EtOH action, in the hippocampal formation, changes in EtOH-sensitive GABAAR s throughout the CNS are likely  or up-regulated  by EtOH exposure. We suggest that the plastic changes in hippocampus are a model for changes in other regions and these could well involve areas/circuits critical for both the dopamine reward system , and for maintained anxiolysis  in the EtOH-dependent individual, rodent or human.Although most measurements were made, justified by region-specific changes in GABAmygdala: –151; venmygdala: , nucleusmygdala: ), thus aAR α2 and γ1 subunit protein in hippocampus, using cross-linking and Western blots. CIE and single-dose EtOH administration up-regulate GABAARs composed of α2β1γ1 subunits that bind to gephyrin, demonstrated by co-immunoprecipitation (co-IP) experiments [, α4, and α5. In contrast to γ2, which was found to associate with different α subunits, γ1 primarily co-assembled with the α2 subunit  are gone; and the withdrawal anxiety is reduced. At 48 h after the 2nd EtOH, all these parameters return to the levels seen 48 h after the 1st EtOH dose: EtOH-sensitive mIPSCs are present; α4 and γ1(α2) subunits are present; withdrawal anxiety is present  and CIE-treated rats  with a decrease in heteropentameric extrasynaptic α4βδ- and synaptic α1βγ2-containing GABAARs and an increase in postsynaptic α4βγ2- and α2β1γ1-containing GABAARs in hippocampal neurons  perfused into the recording chamber potentiated mIPSCs by prolonging decay time and/or increasing charge transfer (area under the curve), as previously observed Liang et al., . For CIEber Fig. , as did ies Fig. . The mIPsynaptic , 165. Pe0 mM perfARs change the kinetics and pharmacological properties of mIPSCs. We previously found decreased diazepam or zolpidem enhancement of mIPSC decay constants and a markedly increased area by the imidazobenzodiazepine partial inverse agonist Ro15–4513 in hippocampal slices after CIE [Up-regulated α2 subtypes correlated with the appearance of synaptic currents enhanced by EtOH (>10 mM). EtOH-enhanced mIPSCs have also been observed in untreated α4KO mice , 145, infter CIE , 139, anfter CIE . These pfter CIE .AR plasticity induced by EtOH in rat hippocampus . This sAR subtypes resulting in hyper-excitability. Adaptations to correct this change do restore inhibition, but it is abnormal, and the animals remain hyperexcitable. Although the EtOH-sedating GABAAR are gone, the replacement GABAARs exhibit EtOH-enhanced synaptic GABAAR inhibitory currents [AR channel blocking convulsant drug PTZ-induced seizures [AR in CIE are the up-regulated α4βγ2 and, especially, α2β1γ1.EtOH induces down-regulation of the first responder receptors, which produces acute tolerance to EtOH, and also triggers the loss of additional GABAcurrents . The CIEseizures , and incseizures , and tolseizures , do not seizures  and presseizures , 124. Alseizures , 109. WeAR-mediated inhibition due to EtOH-induced plasticity of GABAARs. When this becomes persistent due to the CIE treatment, this can be termed ‘aberrant plasticity’ [The behavioral changes of AWS can be explained by persistently reduced GABAsticity’ . The recsticity’ . In the sticity’ , 167. HoAR subunit composition and localization. Acute EtOH induces transient changes in a prescribed temporal sequence, starting with decreased extrasynaptic α4βδ, followed by decreased synaptic α1βγ2 detectable within hours but possibly triggered earlier; about the same time a detectable increase in α4βγ2, including surface expression and synaptic localization, is observed in hippocampus [How do these changes become persistent after EtOH administration that produces a certain number (30~60) of cycles of behavioral depression and hyperexcitable rebound mini-withdrawals?’The CIE animal model exhibits EtOH-induced plastic changes in GABApocampus  and nuclpocampus , as wellpocampus  and basopocampus . The majAR currents in hippocampal slices to determine channel amplitudes, kinetics, and pharmacology, including sensitivity to modulation by EtOH applied in the recording chamber. We have also extended the CIE model to the mouse, and analyzed genetically engineered animals with GABAAR subunits knocked out, in, or down [Attempts to answer this question have included more detailed analysis of the nature and time course of changes in the subunit composition, both total and surface expression, as well as subtype subunit partnering measured by co-immunoprecipitation and Western blotting, including receptor-associated proteins, in hippocampal formation or microdissected DG or CA1. This has been correlated with alcohol intoxication and withdrawal behaviors and patch clamp recordings of GABA or down , 145. Al or down , where cAR [AR synapses after CIE treatment may be abnormal in some way to explain unusual physiology and pharmacology, such as mIPSC kinetics and sensitivity to low millimolar EtOH modulation. This could involve an associated protein, possibly gephyrin/ collybistin [The rapid removal of α4βδ and somewhat slower build-up of α4βγ2 appears to involve de novo synthesis of α4 as well as assembly selectively of α4βγ2 and membrane insertion, plus synaptic localization , not norAR . The regAR  and/or BAR , 171, anAR . We haveAR ) that thlybistin –171, or lybistin . Once folybistin –175.AR expression and surface localization have been demonstrated to be regulated in vivo by a complex region- and cell-specific protein kinase A and protein kinase C system [ARs were prevalent contributors to the mIPSCs in DGC which were enhanced by EtOH in the recording chamber and were rapidly down-regulated by EtOH exposure, unlike in naïve wild type mice or rats, consistent with early internalization of early responder-GABAARs to EtOH in vivo. We also found that the α2-GABAAR subtype of synaptic current that is most abundantly up-regulated in cell surface expression after CIE and that is most sensitive to EtOH modulation in the recording chamber [AR with α2β1γ1 [The α1-GABAC system , 176. InC system , 178. AsC system , we foun chamber . Since m chamber , is comph α2β1γ1 , a rare AR subtypes for a causative role in CIE/AUD, we tested the U. Rudolph α2KO mouse [AR subtypes are candidates of interest in AUD. We mentioned above that evidence suggests that the α2 subunit-containing GABAARs participate functionally in critical neurocircuitry involved in the positive reinforcing effects of EtOH [ARs are needed for the development of EtOH dependence. Increased expression and function might be associated with dependence, and reduced expression and function somehow associated with less susceptibility to developing dependence. This is consistent with genetic association of GABRA2 with alcoholism [Investigating the relevant GABAKO mouse  in the tKO mouse . HoweverKO mouse  showed hKO mouse  and A. HKO mouse . Behavio of EtOH –30, as t of EtOH , 33, and of EtOH . We posicoholism .Fig. 6TwAR in critical brain regions and subcellular membrane locations critical for the anxiolytic response to EtOH and for the elevated drinking in the dependent CIE mouse? If so, it will establish insights into EtOH dependence and drinking and possible therapies that will set the stage for the next generation of questions to answer. For example, how does the EtOH-induced plasticity, namely induction of α2β1γ1 occur, and can it be prevented, or remedied, e.g., with subtype-selective drugs? How do the EtOH-induced plastic changes become persistent, and can that be prevented, or remedied? Is the switch at the level of gene expression or protein trafficking? Where do genetic differences, which we know affect human alcoholism, manifest in such an addiction model? Are the α2β1γ1 and α4 gene cluster SNPs really important? One factor that might be critical for addiction and individual differences is stress [AR-enhancing  neurosteroids (metabolites of progesterone and corticosterone) participate at this level? We are giving a lot of credit to GABAAR plasticity in certain circuits: what about GABAAR changes elsewhere and the behaviors affected  for the receptors involved. Successful therapy for AUD based on GABAAR plasticity would be impetus for more research in the receptor plasticity field.Is the α2-GABAs stress : how impd ? How abAR-PAM drug, including EtOH, neurosteroids [AR down-regulation, compensatory plasticity, producing tolerance and withdrawal, as well as aberrant plasticity involving GABAARs and associated negative effects on behaviors. Neurosteroid GABAAR-PAMs have been demonstrated to produce a hyperexcitable model upon withdrawal, accompanied by anxiety, reduced GABAAR-mediated inhibition, and tolerance to BZs [AR-PAMs [AR plastic changes induced by EtOH [AR PAM neurosteroids [AR plasticity, both by inducing changes in GABAAR expression and localization [AR sensitivity to EtOH [ARs, but this remains an area of interest requiring more research.Administration of any GABAsteroids , benzodisteroids , 89, andsteroids , can inde to BZs , 184, wi[AR-PAMs  may a) a[AR-PAMs ; b) may [AR-PAMs , 137 and by EtOH , 138; c)steroids , and appsteroids , pubertysteroids , pregnansteroids , post-pasteroids , involvilization , and by  to EtOH , 190. It


Prostaglandin D2 (PGD2) and prostaglandin D2 receptor 2 (DP2) is known to be an important factor in androgenetic alopecia (AGA). However, the effect of PGD2 in human dermal papilla cells (hDPCs) is not fully understood. The function of PGD2-induced expression of the androgen receptor (AR), DP2, and AKT (protein kinase B) signal were examined by using real time-PCR (qRT-PCR), western blot analysis, immunocytochemistry (ICC), and siRNA transfection system. PGD2 stimulated AR expression and AKT signaling through DP2. PGD2 stimulated AR related factors (transforming growth factor beta 1 (TGFβ1), Creb, lymphoid enhancer binding factor 1 (LEF1), and insulin-like growth factor 1, (IGF-1)) and AKT signaling (GSK3β and Creb) on the AR expression in hDPCs. However, these factors were down-regulated by DP2 antagonist (TM30089) and AKT inhibitor (LY294002) as well as DP2 knockdown in hDPCs decreased AR expression and AKT signaling. Finally, we confirmed that PGD2 stimulates the expression of AR related target genes, and that AKT and its downstream substrates are involved in AR expression on hDPCs. Taken together, our data suggest that PGD2 promotes AR and AKT signal via DP2 in hDPCs, thus, PGD2 and DP2 signal plays a critical role in AR expression. These findings support the additional explanation for the development of AGA involving PGD2-DP2 in hDPCs. Androgenetic alopecia (AGA) is the most common hair loss disorder in men. AGA is characterized by the replacement of thick terminal hair with fine small vellus hair on the genetic predisposition area of scalp such as frontal and vertex area . The majHistopathologically, inflammatory cell infiltration around the follicular bulge is commonly found in AGA hair follicles . Mahe etCotsarelis and colleagues reported that the levels of prostaglandin D2 synthase (PTGDS) and its catalytic product, prostaglandin D2 (PGD2), were elevated in the balding scalp compared with the non-balding scalp of patients with AGA, as well as PGD2 inhibits mouse and human hair growth through DP2 . The bioTo determine whether DHT affects the PGD2 pathway, human dermal papilla cells (hDPCs) were treated with various doses of DHT for 24 h. Treatment with 100 nM DHT increased cyclooxygenase-2 (COX2), PTGDS and DP2 mRNA expression A–C. MoreTo determine whether PGD2 directly regulates AR expression, hDPCs were stimulated with various concentrations of PGD2 in serum-free medium for 24 h. PGD2 at 50 nM–1000 nM induced the expression of AR. At 200 nM in particular, PGD2 treatment increased the expression of AR (2.3-fold) mRNA at 24 h compared with 0 nM group A. The mRTo investigate the association of the AKT and AR signalling pathways in PGD2-induced hDPCs, hDPCs were treated with PGD2 for different amounts of time, up to 24 h. AKT phsphorylation was observed at 3 and 5 h A. SecondWe confirmed that PGD2-DP2 affects AR expression via AKT and its involved factors . We hypothesized that suppression of DP2 would inactivate AR expression by inhibiting AR-related factors and AKT signalling. Thus, we examined whether inhibition of DP2 could regulate the activity of AR and its related factors. TM30089 has been known as a highly potent antagonist on mouse CRTH2/DP2 . PGD2-inNext, to study the involvement of DP2 in PGD2-induced AR expression, hDPCs were transfected with DP2-targeting siRNA (20 nM). Transfection with DP2 siRNA significantly knocked down the protein level of AR, DP2, COX2 and AKT/GSK3β/Creb phosphorylation, whereas the negative control siRNA (siNC) (20 nM) had no effect A. We alsHuman dermal papilla cells (hDPCs) play an important role in hair follicle formation and hair regeneration and growth . In partDKK-1 and TGFβ1, which are cell death factors, are produced by DHT to destroy hair follicle cells and induce them to enter catagen stage, thereby causing hair loss ,18. ImpoInterestingly, DHT simulated prostaglandin D2 signalling through the expression of COX2, PTGDS, and DP2. Although various stimuli may induce the expression of COX2 in many cells ,20, we uCyclooxygenase-2 (COX2), a pro-inflammatory inducible enzyme, is a key enzyme in prostaglandin (PG) biosynthesis that converts arachidonic acid (AA) to PGG2 and subsequently to PGH2, which is metabolized by various PG synthases to other PGs . PGs areBased on the above results, we performed in vitro analysis to investigate the effect of PGD2 on the expression of AR in hDPCs. Firstly, we investigated the AR signal pathway involved in the effects of PGD2 in hDPCs. We focused on the TGFβ1 and TGFβ2, which involved in hair growth inhibition and apoptosis . We alsoBax (pro-apoptotic gene), while it caused a decrease in the expression of Bcl2 (anti-apoptotic gene) in hDPCs. These results indicate that cell apoptosis was induced by treatment with high concentration of PGD2, indicating the direct involvement of the caspase pathway.Apoptosis is related to the activation of caspases such as caspase-3 and caspase-9. Caspase-9, an initiator caspase, can directly cleave and activate caspase-3 . We founSeveral studies have demonstrated that AKT is involved in signal transduction pathway downstream of a variety of inflammatory mediators, glycogen metabolism and proliferation apoptosis . PhosphoBased on the results of this study, PGD2-induced expression of AR might occur as a result of cell growth inhibition and various genes upregulation because of the activation of DP2. The DP2 antagonist TM30089 is known to regulate the viability of various cell types . Thus, wTaken together, we sought to determine which pathway(s) is critical for the induction of DP2 by PGD2 in hDPCs. Activation of DP2 by PGD2 leads to the AKT signal through G protein-dependent pathway, and more recent results show that activation of the DP2 induces apoptosis through the intrinsic pathway ,34.2. Dihydrotestosterone (DHT) from Sigma-Aldrich . LY294002 (AKT inhibitor) were from Cell Signaling Technology . TM30089 (DP2 antagonist) was from Caymen Chemical . For treatment, the reagents were dissolved in 100% methanol and DMSO to a concentration at 10 mM. Three to fourth-passage DPCs were used in each experiment.Human dermal papilla cells  were purchased from PromoCell . hDPCs were cultured in Follicle Dermal Papilla Cell Growth Media (PromoCell) supplemented with provided mixture reagent at 37 °C in a humidified atmosphere of 5% CO4 cells/well. To test whether DP2 antagonist participate in the viability of PGD2, hDPCs were seeded in a 24-well plate at a density of 1 × 104 cells/well. After 24 h, the medium was replaced with serum-free medium. TM30089 (20 µM) were pretreated with cells for 1 h, and then incubated with or without 200 nM of PGD2 for 72 h. With the addition of 100 µL/well of 3--2,5-diphenyl-2-H-tetrazolium bromide  to each well, the cells were incubated at 37 °C for 4 h. Then, the supernatant was harvested and then treated with 400 µL of dimethyl sulfoxide . The absorbance was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay  reader.hDPCs were seeded in a 24-well plate at a density of 1 × 10Total RNA from the hDPCs using the TRIzolTM reagent  and cDNA synthesis with QuantiTect Rev. Transcription kit  according to the manufacturer’s instructions. The cDNA used for real time-PCR, which was carried out with SYBR Green . The primers sequences and PCR conditions are listed in The protocol for western blot analysis was described in a previous report . Briefly3 cells per well and cultured in serum-free medium in the presence of DHT or vehicle control (methanol) for 5 h. Immunofluorescence staining of AR was performed as previously described [hDPCs were plated in 8-chamber slides  at a density of 2 × 10escribed . Briefly5 cells per 6 well culture dish, washed three times with phosphate-buffered saline (PBS), and then incubated in serum-free medium for 24 h for the collection of conditioned medium. To examine PGD2 receptor induction in response to DHT in hDPCs were treated with varying concentrations of DHT in serum-free medium for 5 or 24 h and concentrations of PGD2 receptor in conditioned medium were measured. Optical density was measured by an ELISA reader at 450 nm.PGD2 receptor ELISA kit  was used according to the manufacturer’s protocol. For the measurement of PGD2 receptor in conditioned medium of DHT-induced hDPCs, cells from passages 3–4 were plated overnight at a density of 2 × 102 incubator until 70–80% confluent. Thereafter, 2 µL DP2 siRNA duplex was diluted into 100 µL of siRNA transfection medium (Santa Cruz Biotechnology). In a separate tube, 2 µL of transfection reagent (Santa Cruz biotechnology) was diluted into 100 µL of siRNA transfection medium. The dilutions were mixed gently and incubated for 30 min at room temperature. Next, cells were incubated in negative control (siNC) or DP2 siRNA transfection cocktail for 5 h at 37 °C. Following transfection, media was changed in all cells to complete media and incubated for a further 18 h. Effects of PGD2 on AR related genes and AKT signaling in normal and DP2-silenced hDPCs were then investigated.Small interfering RNA (siRNA) targeted at DP2 (Santa Cruz Biotechnology) was used to knockout DP2. hDPCs were cultured and incubated at 37 °C in a 5% CO4 cell/well were treated with PGD2 at various concentrations  or DMSO as a control for 72 h. Cell apoptosis was examined with the in situ cell death detection kit  according to the manufacturer’s instructions. The cells were fixed, permeated with 0.1% Triton X-100 solution, labelled for DNA breaks with terminal deoxynucleotide transferase (TdT) dUTP fluorescein nick end labeling  and observed under Axiovert 200 microscope (ZEISS)hDPCs seeded in 24-well plates at a density of 2 × 10p value < 0.05 was considered statistically significant.All data are representative data from three independent experiments. The statistical significance of the differences among groups was tested using one-way ANOVA . All graphs were generated using GraphPad Prism 5 . PGD2 directly stimulates the expression of androgen target genes, AKT and its downstream substrates are involved in mediating these effects. Thus, our data in this study provide that the activity of AR could be regulated not only DHT but also various signal changes by PGD2 in hDPCs.


Bioinformatics and in vitro translation assay were performed to confirm whether or not lnc-PCF is an actual lncRNA. RNA fluorescent in situ hybridization (FISH) and nucleocytoplasmic separation showed that lnc-PCF is mainly expressed in the cytoplasm. Knockdown and knockin of lnc-PCF indicated that lnc-PCF could promote fibrogenesis by regulating the proliferation of epithelial cells activated by TGF-β1 according to the results of xCELLigence real-time cell analysis system, flow cytometry, and western blot analysis. Computational analysis and a dual-luciferase reporter system were used to identify the target gene of miR-344a-5p, whereas RNA pull down, anti-AGO2 RNA immunoprecipitation, and rescue experiments were conducted to confirm the identity of this direct target. Further experiments verified that lnc-PCF promotes the proliferation of activated epithelial cells that were dependent on miR-344a-5p, which exerted its regulatory functions through its target gene map3k11. Finally, adenovirus packaging sh-lnc-PCF was sprayed into rat lung tissues to evaluate the therapeutic effect of lnc-PCF. These findings revealed that lnc-PCF can accelerate pulmonary fibrogenesis by directly targeting miR-344a-5p to regulate map3k11, which may be a potential therapeutic target in IPF.Emerging evidence suggests that microRNA (miRNA) and long noncoding RNA (lncRNA) play important roles in disease development. However, the mechanism underlying mRNA interaction with miRNA and lncRNA in idiopathic pulmonary fibrosis (IPF) remains unknown. This study presents a novel lnc-PCF that promotes the proliferation of TGF- The remaining part of the transcribed genome is known as noncoding RNA (ncRNA). ncRNAs can be divided into small (<200 nt) ncRNAs, such as microRNAs (miRNAs) and transfer RNAs, and long RNAs(>200 nt), such as long noncoding RNAs (lncRNAs) and ribosomal RNAs.4 These lncRNAs generally exhibit the same miRNA response element with the transcripts of mRNAs. For instance, lnc-MD1 ‘sponges’ miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, thereby activating muscle-specific gene expression.5 lnc-APF is identified as the ceRNA that can regulate autophagic cell death by targeting miR-188-3p and ATG7.6 Nevertheless, the mechanism underlying mRNA interaction with miRNA and lncRNA in idiopathic pulmonary fibrosis (IPF) remains unknown. IPF is defined as a specific form of chronic and progressive interstitial pneumonia of an unknown cause, and this disease leads to the progressive loss of lung function, respiratory failure, and death.7 To date, lung transplant is the only effective treatment available for IPF.8 Thus, a comprehensive understanding of the molecular mechanisms of fibrogenesis is required in developing specific therapies toward this disease.9Some lncRNAs regulate the biological function of miRNAs by recruiting the miRNAs as competitive endogenous RNAs (ceRNAs).11 In the present work, we defined a novel lnc-PCF, its function, and the crosstalk between lnc-PCF and the map3k11 target miR-344a-5p in the regulation of fibrogenesis.This paper is a follow-up to a previously completed research in our laboratory. In the previous study, we described the differentially expressed miRNAs, lncRNAs, and mRNAs in IPF; however, the molecular mechanisms of these RNAs are poorly elucidated.11 and we also acquired transcript BC158825, which is significantly different and has higher expression than other transcripts. Therefore, we selected BC158825 for further study and renamed it as lnc-PCF  which revealed its function. According to our database, lnc-PCF is a sense lncRNA located at the second chromosome  with a length of 1208 bp (http://cpc.cbi.pku.edu.cn/) and the coding potential assignment tool (http://lilab.research.bcm.edu/cpat/index.php). The protein-coding ability scores of lnc-PCF were -0.720785 and 0.157507, which indicated that lnc-PCF was devoid of protein-coding potential.14 Lnc-PCF translation activities were measured in an in vitro translation system, which revealed that this gene has no translation activities (in situ hybridization (FISH) and confirmed by quantifying nuclear/cytoplasmic RNA. The results showed that the lnc-PCF transcripts were more localized in the cytoplasm than in the nucleus  is an indicator of pulmonary fibrosis. A significant difference was observed for Hyp level at the given time points compared with that in the sham group -induced animal model by using quantitative real-time PCR (qRT-PCR). The increasing expression level of lnc-PCF was observed at 14, 21, and 28 days, but the level decreased at 7days , which iam group . The higam group .17 TGF-β1 was used to stimulate the rat lung epithelial-t-antigen negative (RLE-6TN) cells to confirm the results in vitro. Lnc-PCF was upregulated in TGF-β1-induced RLE-6TN cell model as compared with that in the normal group , which targeted six different sections of lnc-PCF, and the recombinant plasmid (pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS) of the overexpressed lnc-PCF  were designed and transfected to RLE-6TN cells to investigate and confirm the function of lnc-PCF in pulmonary fibrosis. The efficiency of knockdown and knockin lnc-PCF was subsequently evaluated by qRT-PCR. The lnc-PCF smart silencer effectively interfered with lnc-PCF expression . Meanwhiα-smooth muscle actin (α-SMA), vimentin, transcription repressor Snail, and loss of the epithelial cell marker E-cadherin.19 The protein levels of these markers were examined after incubation with TGF-β1 for 72 h under knockdown or knockin lnc-PCF by using immunofluorescence staining and western blot analysis to investigate the effect of lnc-PCF on fibrogenesis. Immunofluorescence data showed that the α-SMA level was significantly higher in the cells treated with TGF-β1 alone than in the other groups. Nonetheless, α-SMA expression reached lower levels in the cells treated with lnc-PCF smart silencer +TGF-β1 than in TGF-β1+NC group  in a 2D structure that contains the binding site position of 3′-UTR or full-length sequence of lncRNA, miRNA, and lnc-PCF should be completely paired. The binding type includes 8mer, 7mer-m8, 7mer-al, 6mer, offset 6mer, and imperfect . Except for imperfect type, other types possess approximately the total number of paired nucleotides. Seed match is not required if the binding type is imperfect. (2) Many red bars that mark the AU weight located on both sides of the seed sequence are preferable. (3) Binding sites that are close to both sides are suitable. MiR-344a-5p, miR-138-5p, miR-370-3p, and miR-484 were selected for the study according to their relative high affinity between lnc-PCF and miRNAs . These min vitro , therebyRenilla. The luciferase genetic testing report showed that miR-344a-5p overexpression suppressed the luciferase activity of the WT reporter vector but not that of the mutant reporter vector  and mutant-type (MT) 3′-UTR, which was behind two luciferases, namely, firefly and r vector . This rein vitro. qPCR analysis revealed that lnc-PCF was significantly enriched in miR-344a-5p as compared with that in non-targeting miR-708-3p (β1-treated RLE-6TN cells. The miR-344a-5p overexpression (mimic) resulted in decreased lnc-PCF expression, and the suppression of miR-344a-5p (inhibitor) significantly enhanced the lnc-PCF expression as compared with that in the TGF-β1+NC group , and rescue experiments were conducted to further verify the direct targeted relationship between lnc-PCF and miR-344a-5p. RNA pull-down was performed to detect the endogenous miR-344a-5p associated with lnc-PCF using transcribed biotin-labeled lnc-PCF R-708-3p . MoreoveR-708-3p . To inveNC group . In addiNC group  but was NC group . All theα-SMA, vimentin, and Snail, which were promoted by lnc-PCF overexpression. All of these findings revealed that lnc-PCF promotes cell proliferation that is dependent on miR-344a-5p by directly targeting miR-344a-5p.Given that lnc-PCF was confirmed to directly target miR-344a-5p, experiments were designed to verify whether or not lnc-PCF promotes the proliferation of activated epithelial cells dependent on miR-344a-5p. The miR-344a-5p mimic and inhibitor were transfected into RLE-6TN cells. Cell proliferation profiles were determined by flow cytometry, western blot analysis, and real-time cell analysis system. Data showed that due to the upregulated miR-344a-5p (mimic) expression, the cells accumulated in G0/G1, and the percentage of cell accumulation in the S and G2/M phases is lower than that in the control . Western24miRNAs exert their regulatory functions through specific interactions with their target genes. Therefore, the miR-344a-5p target genes were predicted based on TargetScan, miRanda data, and miRbase. The affinity of predicted target genes with miR-344a-5p was also analyzed. Among the list of miR-344a-5p target genes, we focused on map3k11 because it has relatively higher affinity than other genes and is an important indicator of the master regulatory factor in cell differentiation, proliferation, and individual development.β1 to further confirm the target relation between map3k11 and miR-344a-5p. Data showed that the miR-344a-5p mimic could inhibit map3k11 expression, whereas the miR-344a-5p inhibitor could promote map3k11 expression as compared with that in the TGF-β1-activated group . The rats in the sh-lnc-PCF group presented a continuous bronchial mucous membrane structure with a more intact wall than those in the BLM group. The alveoli in the sh-lnc-PCF group showed clearer hollow cavities with thinner alveolar walls than those in the BLM group. In addition, the lung mesenchyme in the sh-lnc-PCF group displayed few collagen fibers, thereby indicating that the hallmark of the fibroblastic foci was distinctly decreased (An interfered sequence of lnc-PCF(sh-lnc-PCF) was synthesized, packaged in adenovirus, and sprayed across rat lung tissues by using a Penn-Century MicroSprayer  to determine the potential of lnc-PCF as a therapeutic target ecreased . The BLMecreased .in vivo, we tested the expression levels of E-cadherin, SP-C, α-SMA, collagen III, and vimentin, which are indicators of pulmonary fibrosis, after sh-lnc-PCF spraying in vivo. The sh-lnc-PCF could decrease the expression levels of a-SMA, collagen III, and vimentin, but increase those of E-cadherin and SP-C in the sh-lnc-PCF group as compared with those in the BLM group  and maintained according to the regulation approved by the Institutional Animal Ethics Committee of Binzhou Medical University.The interfered sequence of lnc-PCF was packaged in the adenovirus. The treatment of adenovirus packaging interfered with the sequence of lnc-PCF was as follows: the rats were randomly divided into the following four groups (10 rats each): sham, BLM-treated (BLM), BLM+empty vector, and BLM+sh-lnc-PCF. The adenovirus packaging interfered with lnc-PCF sequence was sprayed into the rat lung tissues by using a Penn-Century MicroSprayer (Penn-Century Inc.). All rats were killed on day 28. Lung tissue sections were collected and immediately frozen in liquid nitrogen for further studies.2 atmosphere. This cell line was further used for experiments upon reaching 70–80%.RLE-6TN cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences  and maintained in DMEM medium  supplemented with 10% fetal bovine serum and 2% penicillin and streptomycin under the condition of 37 °C and 5% CO5 cells per well) until the cell density reached 70–80% prior to small RNA transfection. On the following day, the cells were transfected with 50 nM small RNA accompanied with reagent buffer by using a ribo FECT™CP Transfection Kit  according to manufacturer’s instructions. After 6 h of culture at 37 °C, the complexes were replaced with complete medium. All cells were collected for Western blot or other experiments.Lnc-PCF smart silencer, miR-344-5p mimic, and miR-344-5p inhibitor were purchased from RiboBio Co., Ltd. . The RLE-6TN cells were plated in six-well culture dishes  on a Rotor Gene 3000 RT-PCR system of Corbett Research . The PCR reaction system for miRNA was as follows: initial denaturation at 95 °C for 20 s and30 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 10 s. For lncRNA, the reaction system was as follows: initial denaturation at 95 °C for 30 s and 35 cycles of 95 °C for 5 s and 60 °C for 20 s. The threshold cycle was determined after the reactions. The relative miRNA and lncRNA expression levels were calculated based on Ct values and were normalized to the U6 or GAPDH levels of each sample, respectively.Total RNA from collected cells was extracted using Trizol reagent. The growth media were briefly removed from the culture dish. Approximately 1 ml of TRIzol was directly added to each 9.5 cm5 RLE-6TN cells were cultivated on the sterile slides in a 24-well plate. After small RNA transfection and TGF-β1 treatment, these cells were rinsed with cold phosphate-buffered saline (PBS) for three times and fixed in 4% paraformaldehyde for 1 h. The cell lines were rinsed with PBS for three times, incubated with 0.5% Triton X-100 for 15 min at room temperature, and blocked with 10% normal goat serum for 1 h at 37 °C. Afterward, α-SMA antibody (1 : 300) was added dropwise into each slide at 4 °C overnight. RLE-6TN was rinsed with PBS for three times and incubated with fluorescein-labeled immunoglobulin G (IgG) antibody (1 : 200) α-SMA (FITC-labeled) at 37 °C for 1 h in a wet box. All operating steps were conducted in the dark because fluorescent secondary antibodies were added. After rinsing with PBS for three times, the nuclei were stained with DAPI  for 5 min at room temperature. Immunofluorescence was analyzed under a fluorescence microscope.Approximately 2 × 10μg of protein was run on a 10% SDS-polyacrylamide gel. After being electrophoretically transferred to a pure nitrocellulose blotting membrane , the bolts were probed with primary antibodies, with GAPDH as an internal control. Exposure was performed using a chemiluminescence imaging system . The bands were quantified by measuring the band intensity for each group.Cells were lysed with PIPA lysis buffer. A total of 30 5 cells were collected and resuspended in 500 μl of 70% cold ethanol, which was added dropwise under gentle vortexing. The cells were fixed overnight at 4 °C, collected by centrifugation, washed once in PBS, collected again, and stained with propidium iodide (PI) staining solution, which was composed of 20 μg/ml PI, 200 μg/ml RNAse A, and 0.1% Triton X-100 for 15 min at 37 °C. The DNA contents of the stained cells were analyzed using a flow cytometer.Approximately 1 × 10MluI and HindIII, were used to sever the lnc-PCF sequence at AAGCTT and ACGCGT, respectively. The digested lnc-PCF was inserted into the rear of the luciferase reporter genes (firefly and Renilla) in Pmir-REPORT-lncPCF pronucleus Amp resistance. Plasmid-transfected 293 T cells: 293 T cells were cultivated in a 96-well plate at a density of 2 × 105/well. After 24 h, lnc-PCF and transfection reagents were prepared using 0.2 μg of firefly, 0.01 μg of Renilla, and 0.25 μl of transfection reagents and were incubated for 5 min. MiR-344a-5p transfection reagents were prepared at the final density of 100 nM miRNA and 0.25 μl of transfection regents and incubated for 5 min. Afterward, we mixed lnc-PCF and miR-344a-5p with their transfection reagents and incubated the mixtures for 20 min. About 50 μl of the medium was removed, and 25 μl of lnc-PCF transfection mixture and 25 μl of miR-344a-5p transfection mixture were added to the cultured cells. After transfection for 6 h, the transfection mixture was replaced with a new complete medium. Dual-luciferase reporter assay: after cotransfection for 48 h, the complete medium was removed, and the cells were rinsed using 100 μl of PBS, which was subsequently replaced with 1 × passive lysis buffer to lyse the cells. The mixture was shaken for 15 min. Each well was added with 100 μl of Stop&Gloreagent for 2 s to obtain test data under dark condition.Vector construction: we used the forward-sequenced primer (5′-GAGGAGTTGTGTTTGTGGAC-3′) and reverse-sequenced primer (5′-TGTAAAACGACGGCCAGT-3′) to obtain the lnc-PCF sequence. Restriction enzymes, namely, μl of restriction endonuclease was added to 20 μg of plasmid. The total volume was adjusted to 50 μl by using H2O. The mixture was incubated at 37 °C from 2 h to overnight. The linearized plasmid was purified using the QIAquick Gel Purification Kit. After purification and size check of RNA, the prepared denatured RNA gel was used to confirm whether or not the RNAs were transcribed at the right size. Sufficient cells were cultured and collected at a minimum of 10 cm × 10 cm plate for each pull down. The cell pellet was suspended using 1 mL of ProteaPrepZwitterionic cell lysis buffer supplemented with protease and phosphatase inhibitor cocktails (1 : 100), anti-RNase (1U/μl), panobinostat (1 : 100), and methylstat (1 : 100). The tube was then incubated on ice for 40 min and vortexed every 10 min. The mixture was centrifuged at 14 000 × g for 15 min at 4 °C, and the supernatant was transferred to a new tube for further use. Afterward, 50 μl of beads were transferred to a fresh tube. The tube was placed on a magnetic separator for 1 min, and the supernatant was removed. After clearing the lysate with activated beads to reduce nonspecific binding, 20 μg of biotinylated RNA was heated to 90 °C for 2 min. The mixture was chilled on ice for 2 min and briefly centrifuged. The total volume was adjusted to 100 μl by using the RNA structure buffer. The mixture was incubated at room temperature for 20 min to allow proper secondary structure formation. About 50 μl of activated avidin magnetic beads were also prepared. The beads were immediately subjected to RNA (20 μg) capture in RNA capture buffer for 30 min at room temperature with gentle agitation. The RNA capture buffer was removed. The RNA-captured beads were washed once with NT2 buffer and incubated with 30 mg pre-cleared cell lysate for 2 h at 4 °C with gentle rotation. The tube was centrifuged and placed on a magnetic separator. The supernatant was removed from the beads and discarded. The RNA-binding protein complexes were washed with NT2 buffer twice, NT2 high-salt buffer (500 mM NaCl) twice, NT2 high-saltbuffer (1 M NaCl) once, NT2 high-saltbuffer (750 mM KSCN) once, and PBS twice. The beads were eluted by incubating the sample with elution buffer for 20 min at 4 °C with frequent agitation, and the elute was collected. The elution step was repeated, and the elutes were pooled together. PCR was used to test the samples.Cloning of the lnc-PCF sequence into Pgen-3z vector: the lnc-PCF sequence was ligated into multiple cloning sites located between the T7 promoter (5′-TAATACGACTCACTATAGGG-3′) and SP6 promoter (5′-AATTTAGGTGACACTATAGAA-3′). Approximately 5 RIP experiment was performed using an EZ Magna RIP kit  following the manufacturer’s protocol. The cells were lysed in complete RIP lysis buffer. The extract was incubated with Ago2 antibody or control IgG  6 h at 4 °C, followed by adding magnetic beads conjugated with proteinA/G . The beads were washed and incubated with Proteinase K to remove the proteins. Finally, purified RNA was subjected to qRT-PCR analysis using specific primers for lnc-PCF.2O in this work. This reaction allowed the measurement of any background incorporation of labeled amino acids. T7 Luciferase Control DNA was used as positive control. The reaction was incubated at 30 °C for 60–90 min, and the translation results were analyzed. After SDS-PAGE electrophoresis, the gel was stained using InstantBlue Ultrafast Protein Staining Solution .The T7-BC158825 sample DNA was prepared by PCR using the following primers: T7-T7-BC158825-F:5′-ACCGCCTAATACGACTCACTATAGGGACCTCCACCTGCATGACCCTGGC-3′T7-BC158825-R:5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTGAGTGAATAAATGAGCCAATAT-3′. The PCR product was purified using a Gel Extraction Kit . The reagents were removed from storage at −70 °C. The TnT Quick Master Mix  was rapidly thawed by hand-warming and placing on ice. Other components were thawed at room temperature and stored on ice. Following the component chart above, we assembled the reaction components in a 0.5 or 1.5 ml microcentrifuge tube. When all of the components were added, the mixture was gently mixed by pipetting. If necessary, the mixture was centrifuged briefly to return the reaction to the bottom of the tube. We included a control reaction with no added DNA, that is, Ht-test was used to compare the two groups. One-way ANOVA with Student–Newman–Keuls post hoc test was conducted to compare three or more groups. Statistical significance was considered at P<0.05.Data were statistically analyzed in SPSS version 19.0 . Data were presented as mean±S.D. of at least three independent experiments. Unpaired Student’s Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Agastache rugosa (Fisch. & C.A. Mey.) Kuntze (A. rugosa) has been used to treat nausea, vomiting and dispel damp. However, currently, few reports about the chemical constituents, especially the non-volatile components of A. rugosa are available.In the Chinese traditional medicine, plant of A. rugosa, the biological activities of the major constituents were investigated. The extracts and main constituents of A. rugosa were evaluated for their anticoagulant effects by assaying the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) in vitro.Through separation with various column chromatographies to elucidate the chemical constituents of 1–7) were isolated from the aerial parts of A. rugosa. They were identified as methyl hexadecanoate (1), β-sitosterol (2), acacetin (3), ursolic acid (4), apigenin (5), protocatechuic acid (6) and tilianin (7), respectively. Compounds 1 and 6 were isolated from the genus Agastache for the first time, and compound 4 was obtained from the plants for the first time. The results showed that the extract of A. rugosa had a significant procoagulant activity by shortening the time of PT (P < 0.001) and increasing FIB content (P < 0.001), as compared with Vitamin K1. While its major constituents acacetin and tilianin exhibited significant anticoagulant activities by prolonging the times of PT, APTT, TT and reducing FIB content (P < 0.001), as compared with blank control group.Seven known compounds (namely compounds A. rugosa possessed significant procoagulant activity, while its main components, acacetin and tilianin possessed significant anticoagulant activities. Further investigation should be pursued to find out the bioactivity components responsible for the procoagulant action of the plant.The total extract of  Agastache rugosa (A. rugosa), a medicinal plant belonging to the family Lamiaceae, is native to Korea, Japan, and China. A. rugosa shows the taste and thermal properties as pungent (acrid) is slightly warm and can the channel affiliations to enter spleen, stomach and lung. A. rugosa has been used in folk medicine to treat a variety of diseases, including cholera, vomiting, miasma and other intestinal disorders +. 1H-NMR  δ: 0.87 , 1.25 , 1.61 , 2.29 , 3.66 . 13C-NMR  δ: 174.2 (C-1), 51.35 (C-1′), 34.11 (C-2′), 24.96 (C-3), 29.15 (C-4), 29.24 (C-5), 29.34 (C-6), 29.67 , 29.58 (C-12), 29.44 (C-13), 31.91 (C-14), 22.67 (C-15), 14.06 (C-16). The above spectral data were basically consistent with those reported previously [1 was identified as methyl hexadecanoate.Compound 2: this compound was a white powder with mp at 136–137 °C. The molecular formula was determined to be C29H50O. EI-MS m/z: 414[M]+. When compound 2 was compared with reference substance of β-sitosterol, no difference was seen between them in term of the TLC detection. Its Rf values were the same in three different developing agents, and the coloration were the same. The samples were mixed with reference substance of β-sitosterol, and the melting point of the mixture was not decreased. Thus compound 2 was identified as β-sitosterol.Compound 3: this compound is a yellow, needle-shaped crystal with mp at 261–262 °C. The molecular formula was determined to be C16H12O5. EI-MS m/z: 283 [M-H]−. 1H-NMR  δ: 12.88 , 8.01 , 7.11 , 6.82 , 6.50 , 6.20 , 3.84 . 13C-NMR  δ: 122.98 (C-1), 163.61 (C-2), 103.67 (C-3), 181.97 (C-4), 157.57 (C-5), 94.29 (C-6), 164.45 (C-7), 99.14 (C-8), 162.54 (C-9), 103.94 (C-10), 161.61 (C-4′), 128.52 , 114.82 , 55.75 (4′-OCH3). The above data were basically consistent with those reported in the reference [3 was identified as acacetin.Compound 4: this compound was a white crystalline with mp at 240–245 °C. The molecular formula was determined to be C30H48O3. EI-MS m/z: 456[M]+. 1H-NMR  δ: 5.50 , 3.46 , 2.33 , 1.25 , 1.24 , 1.06 , 0.95 , 0.90 , 1.02 , 1.03 . 13C-NMR  δ: 37.17 (C-1), 27.85 (C-2), 77.88 (C-3), 38.84 (C-4), 55.57 (C-5), 18.52 (C-6), 33.32 (C-7), 39.73 (C-8), 47.78 (C-9), 39.23 (C-10), 23.64 (C-11), 125.38 (C-12), 138.99 (C-13), 42.25 (C-14), 28.53 (C-15), 24.65 (C-16), 47.78 (C-17), 53.30 (C-18), 39.14 (C-19), 39.09 (C-20), 30.81 (C-21), 37.03 (C-22), 28.43 (C-23), 15.40 (C-24), 16.27 (C-25), 17.23(C-26), 23.36 (C-27), 179.55 (C-28), 17.18 (C-29), 21.11 (C-30). The above data were basically consistent with those reported in the reference −. 1H-NMR  δ: 7.33 , 7.29 , 6.79 . 13C-NMR  δ: 121.87 (C-1), 116.74 (C-2), 145.03 (C-3), 150.15 (C-4), 115.31 (C-5), 122.03 (C-6), 167.43 (C-7). The above data were basically consistent with those reported in the reference [6 was identified as protocatechuic acid.Compound 7: this compound was a light yellow powder with mp at 256–257 °C. The molecular formula was determined to be C22H22O10. EI-MS m/z: 446[M]+. 1H-NMR  δ: 12.91 , 8.07 , 7.14 , 6.95 , 6.86 , 6.46 , 5.06 , 3.71 , 3.18 , 3.45 , 3.47 . 13C-NMR  δ: 163.00 (C-2), 103.78 (C-3), 181.98 (C-4), 156.93 (C-5), 99.56 (C-6), 163.80 (C-7), 94.90 (C-8), 162.44 (C-9), 105.36 (C-10), 122.66 (C-1′), 128.39 , 161.07 (C-4′), 114.59 , 55.54 (4′-OCH3), 99.94 (C-2′′), 73.08 (C-2′′), 77.15 (C-3′′), 69.57 (C-4′′), 76.41 (C-5′′), 60.60 (C-6′′). The above data were basically consistent with those reported in the reference [7 was identified as tilianin. All the sructures of compound 1 ~ 7 were shown in Fig. Compound eference . Thus, cP < 0.001) while Ar.Pe, Ar.Ea, and Ar.Bu had highly significant effects on promoting blood coagulation (P < 0.001). Compared with the breviscapine, the effects of acacetin and tilianin were not better than that of the positive control (P < 0.001). Compared with the Vitamin K1, Ar.Pe and Ar.Bu had significant effects on promoting coagulation .In Fig. P < 0.001) while Ar.TE, Ar.Pe, Ar.Ea, Ar.Bu had highly significant effects on promoting blood coagulation . Compared with the breviscapine, the effects of acacetin and tilianin were not better than the positive control (P < 0.001). Compared with the Vitamin K1, Ar.TE and Ar.Pe had significant effects on promoting coagulation .Results shown in the Fig. P < 0.001). Compared with the breviscapine, the anticoagulant activity of the positive control was the best one (P < 0.001).As shown in Fig. P < 0.001). Compared with the Vitamin K1, Ar.TE could significantly increase FIB content (P < 0.001).Results shown in the Fig. A. rugosa. These compounds have been known to have a wide range of biological activities and play a role in triggering cardiovascular activities. For instance, Li et al. [+ channels, both could cause the antihypertensive effect [Through the chemical analysis, we found that ursolic acid, acacetin and tilianin were the main chemical components in i et al.  reportedi et al.  reportedi et al.  demonstre effect .A. rugosa. In this study, we found that the main chemical components, including ursolic acid, acacetin and tilianin of A. rugosa had anticoagulant-related activity. Anticoagulant activities of Ar.TE, Ar.Pe, Ar.Ea, Ar.Bu, acacetin and tilianin were investigated in the experiment in vitro.There have been no reports in the literature about the anticoagulant activity of The results obtained in this study are quite interesting. Firstly, Ar.TE had an activity of promoting coagulation as shown by PT and FIB while Ar.Pe, Ar.Ea and Ar.Bu had procoagulant activities as shown by APTT and PT. Acacetin, ursolic acid and tilianin all displayed significant anticoagulant activities as shown by PT, APTT, TT and FIB. The activities of Ar.Pe, Ar.Ea and Ar.Bu were different because their main components were different. For instance, acacetin and ursolic acid were the main components in the Ar.Ea while tilianin was one of the main components in the Ar.Bu. The difference in main components might be due to following reasons: the compounds having a great effect might not be isolated or the remaining compounds could have a great effect on promoting coagulant, though their contents might be very low. The chemical components of plant medicines are complex, and the pharmacological effects can be the interactions of multi-components, multi targets together, either synergistically or antagonistically. There might be no any compounds having great effects on promoting coagulation. This is the common function of several components.50 = 933 mg/kg) [As compared with the blank group, both acacetin and tilianin exhibited significant anticoagulant activities as demonstrated by PT, APTT, TT and FIB assays. Acacetin and tilianin are natural flavonoid compounds with abundant sources from plant origin. The techniques for isolation and separation of both compounds are simple and easy to operate. The acute toxicity experiment of acacetin showed that it was a safe clinical medication (LD3 mg/kg) . The acuA. rugosa has been commonly used to treat summer heat-dampness and stomach flu etc. Because A. rugosa is also an aromatic plant, the studies on A. rugosa have been mostly focused on the volatile components. The research on non volatile component of A. rugosa was mainly initiated in the 1990s [A. rugosa. Thus, we investigated the chemical composition and coagulation activity of A. rugosa and found that Ar.TE had a significant procoagulant activity, compared with Vitamin K1. Acacetin and tilianin had significant anticoagulant activity, as compared with the blank group. Further investigation should be pursued to find the effect of A. rugosa, acacetin and tilianin in vivo. Since the effects of acacetin and tilianin are different from those of A. rugosa, it should be noted that other active components in the extract remained to be further identified and characterized.he 1990s –30, and A. rugosa has been a traditional Chinese medicinal plant. The previous studies on A. rugosa were mainly focused on the volatile components and its traditional pharmacological activity. In the present study, we found for the first time that the A. rugosa had a significant coagulant activity. The total extract of A. rugosa possessed significant procoagulant activity while acacetin and tilianin identified as the main components of A. rugosa had a significant anticoagulant activity in vitro. As a novel, effective and promising drug for the treatment of various coagulation disorders, A. rugosa may be beneficial for the individual with high risks of with hemophilia and other bleeding disorders. Acacetin and tilianin were natural flavonoid compounds, whose plant origin was abundant and isolation and separation technique were simple and easy to operate. They might have a good pharmaceutical prospect. Being cheap, safe and effective, they can meet requirements of today’s market about antithrombotic drugs.


Hyperglycemic hemichorea tends to affect elderly patients with type 2 diabetes, women, and the Asian population. The onset of involuntary movement typically occurs at the hyperglycemic state and subsides at the euglycemic state. In this report, we present an unusual case that developed delayed-onset hemichorea after hyperglycemia correction.A 70-year-old man was admitted to neurology ward with symptoms of subacute dizziness. Hyperglycemia and high level ketone body was incidentally noted. Hemichorea occurred in his left limbs 2 days after hyperglycemia correction.Patient remained conscious, and no other focal neurological deficits were noted while hemichorea occurred. Blood test revealed no contributory cause. Brain magnetic resonance imaging revealed no lesions in the putamen or subthalamus. A diagnosis of probable hyperglycemia-related hemichorea was made.Haloperidol  was prescribed.Hemichorea improved gradually before discharge and resolved 4 months later.Differential diagnosis of hemichorea should include delayed-onset hemichorea after hyperglycemia correction. Here, we report a rare case of acute hemichorea that occurred after hyperglycemia correction.Hemichorea is an uncommon but remarkable neurological disorder associated with hyperglycemia. Elderly patients with type 2 diabetes, women, and the Asian population have a higher risk of hyperglycemic hemichorea.22O), and blood ketone bodies (5.9 mmol/L). Renal functions, hepatic functions, and blood gas were within the normal range. Brain computed tomography performed at the emergency department revealed negative result. Subsequently, the patient was admitted, and oral hypoglycemic agents including Metformin (1000 mg/d) and Glimepiride (2 mg/d) were prescribed; intermittent subcutaneous insulin was administered if the blood sugar level increased >200 mg/dL. After 2 days, acute hemichorea was observed in his left limbs; the blood sugar level was 168 mg/dL. The blood sugar level did not increase during these 2 days. He remained conscious, and no other focal neurological deficits were noted. Other laboratory test results were within the normal range, including the blood cell count, renal function, liver function, thyroid function, vitamin B12 level, autoimmune profile, human immunodeficiency virus antibody titer, and rapid plasma reagin. Brain magnetic resonance imaging (MRI) performed after hemichorea onset showed no infarction or hyperintensity lesion in the right basal ganglion and subthalamus , glycated hemoglobin (19%), osmolality . However, because of the prolonged interval between hyperglycemia and onset of hemichorea, the relationship between them remains debatable. Taboada et al reported a case of an elderly female patient with transient ischemic attack and hyperglycemia (blood sugar level: 700 mg/dL) at the initial presentation. Hemichorea occurred 1 week after partial hyperglycemia correction (blood sugar level: 486 mg/dL). However, the blood sugar level did not return to its normal range during hemichorea onset.Generally, the blood sugar level is extremely high during hemichorea onset. However, in this case report, we present an unusual disease course, in which the involuntary movement occurred after hyperglycemia correction. In the literature, 2 cases of delayed-onset hemichorea after hyperglycemia correction have been reported. Bizet et al However, the association between neuroimaging findings and symptoms varied. Some cases had no typical neuroimaging lesions in the basal ganglion, whereas others had bilateral neurological lesions associated with hemichorea.,4 The pathological changes in putaminal lesions remain unclear. Advanced neuroimaging studies, such as magnetic resonance spectroscopy (MRS), single-photon emission computed tomography (SPECT), and 2-[18F] fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET), have provided some possible explanations. MRS revealed relatively low N-acetylaspartate to creatine ratio, high choline to creatine ratio, and lactate peak, indicating neuronal damage, gliosis, and acute or chronic ischemic changes, respectively. Furthermore, SPECT suggested hypoperfusion in the striatum, and FDG-PET demonstrated reduced rates of cerebral glucose metabolism in the corresponding areas with abnormalities on T1-weighted MRI.The most commonly reported neurological image findings among the hyperglycemic hemichorea cases were lesions in the putamen contralateral to the affected limbs.,8 Second, vasculopathy was hypothesized to be a possible mechanism. Long-term hyperglycemia causes hyperviscosity of blood, which leads to latent ischemia of the striatum and subsequent dyskinesia. Although this hypothesis was not confirmed in most cases with unilateral lesions, it was partially supported by a striatal biopsy after hyperglycemia-related hemichorea, which showed neuron loss, gliosis, and reactive astrocytosis. Ketone bodies may play a role in hyperglycemia-related hemichorea. The absence of insulin leads to hyperglycemia and the release of free fatty acids from the adipose tissue, which are metabolized to ketone bodies (acetoacetateand β-hydroxybutyrate). Acetoacetatecan can be used as a GABA substitute to temporarily compensate for the hyperglycemia-induced GABA depletion. Thus, this finding reflects the rarity of hemichorea in patients with diabetic ketoacidosis.The molecular pathogenesis underlying hyperglycemic hemichorea remains unclear, and 2 major hypotheses have been proposed. First, γ-aminobutyric acid (GABA) was used as an alternate energy substrate during hyperglycemic crisis. GABA depletion leads to thalamic disinhibition and hyperkinesia.,3 We are the first one who mentioned ketone bodies play a role in this condition, which stress the need for further studies to prove this hypothesis. Second, in a small review for hemichorea in ketotic hyperglycemia, no patient presented with delayed hemichorea after hyperglycemia correction.Our patient demonstrated a remissive course of involuntary movement and in absence of putaminal abnormalities on MRI, which support the hypothesis that hyperglycemia self plays a major pathogenetic role. Delayed chorea may due to temporarily compensate for the GABA depletion by high level ketone bodies initially. However, our hypothesis has some limitations. First, ketone bodies level was not mentioned in the previous case reports for delayed hyperglycemia-related chorea.4Although a rare possibility, differential diagnosis of hemichorea should include delayed-onset hemichorea after hyperglycemia correction. We hypothesize that hyperglycemia-induced ketone bodies play a crucial role in delayed-onset hemichorea; however, the underlying molecular pathogenesis remains unclear.The authors acknowledge Wallace Academic Editing for editing this manuscript.


Androgen receptor (AR) is a promising therapeutic target for breast cancer. However, its prognostic value remains controversial in triple negative breast cancer (TNBC). Here we present a meta-analysis to investigate the correlation between AR expression and TNBC prognosis.p< 0.001), but more lymph node metastases (p < 0.01). AR positivity was associated with prolonged disease free survival , but had no significant impact on overall survival . No difference in survival existed between subgroups using different AR or estrogen receptor cutoff values.Thirteen relevant studies with 2826 TNBC patients were included. AR positive rate was 24.4%. AR+ patients tended to have lower tumor grade (Literature search was performed in Pubmed, Embase and Cochrane Central Register of Controlled Trials databases to identify relevant articles on AR and TNBC prognosis. Fixed- and random-effect meta-analyses were conducted based on the heterogeneity of included studies. Heterogeneity and impacts of covariates were further evaluated by subgroup analyses and meta-regression.AR positivity is associated with lower risk of disease recurrence in TNBC. Further clinical studies are warranted to clarify its prognostic role on TNBC recurrence and survival. There w= 0.270) .p < 0.01, I-square = 58.9%). By random-effect meta-analysis, AR positivity was not associated with any improvement in OS   , study population , ethnicity (p = 0.054), ER cutoffs  or statistical analysis   nor high cutoff 5 or 10%) subgroup revealed AR-related benefit for OS  Figure . There w= 0.165) . Removal= 0.165) .Meta-regression was performed on outcome data. None of the covariates showed statistically significant effects on DFS or OS .p = 0.537, OS p = 0.945).Funnel plots Figure  for DFS As a novel therapeutic target, AR was widely investigated in many pre-clinical and clinical studies for TNBC , 19, 26.et al. found that AR+ was beneficial to DFS in TNBC subgroup , but not for OS  . St. St27]. 61-1.32) . Kim et ctively) , 23, 24,et al. which demonstrated AR expression incurred better prognosis irrespective of ER co-expression [Several studies revealed the antagonizing effect of AR against ER signaling depended on the relative levels of these two steroids receptors. They competed for co-regulatory molecules, resulting in opposite effects on cancer cell proliferation . Withoutpression . With ERSubgroup analyses showed remarkable decrease in the heterogeneity of OS in subgroups of whole-cohort TNBC patients and high AR/ER cutoffs , suggestOur studies had several limitations. First, it based on population data other than individual patient data, and restrained our ability to conduct analyses for LN metastases and other covariates. Second, all the studies were retrospective. It could potentially increase certain bias, such as selection bias. Third, we were unable to identify correlation between AR and molecular intrinsic subtypes of TNBC, especially for LAR subtype which may be helpful to clarify AR prognostic value.In conclusion, although variability and heterogeneity existed among included studies, AR expression was associated with lower risk of disease recurrence. Since AR had a relatively high prevalence in TNBC, evaluating AR status could provide additional information on prognosis and AR-targeting therapy could be another option for chemotherapy resistant TNBC in both neoadjuvant and metastatic settings. Future clinical trials based on different molecular subtypes of TNBC are warranted to further clarify its prognostic value and efficacy of AR-targeting therapy.We searched the following databases for relevant studies: PubMed , Embase  and Cochrane Central Register of Controlled Trials . The following medical subject headings, Embase Emtree terms and keywords were used: ‘Breast Neoplasms’, ‘Triple Negative Breast Neoplasms’, ‘Breast Cancer’, ‘Triple Negative Breast Cancer’, and ‘Androgen Receptor’. There was no limitation on languages or regions of publications. Reference lists of all the relevant articles were manually screened to ensure the sensitivity of the literature search.et al. [To be eligible, studies had to meet the following inclusion criteria: TNBC patients, or studies that provided data on TNBC as a subgroup; assessment of AR expression in primary breast cancer tissue; available data of OS or DFS on patient subgroups with different AR level. Exclusion criteria included: metastatic disease; review, meta-analysis, editorial, letter or conference abstract. Two independent reviewers (C.J. Wang and B. Pan) evaluated eligibility of studies according to the above criteria. Full text of the potentially relevant studies were obtained and reviewed for inclusion by the same two reviewers. Disagreement was resolved by consensus . Inter-reviewer agreement was assessed by Kappa Statistics according to Higgins et al. The quality of the included studies was assessed according to the STROBE checklist for Cohort Studies . Each itp-value) were extracted from tables or text of included studies, or estimated from Kaplan-Meier curves where applicable [Data were collected using a predesigned data extraction form by two reviewers (C.J Wang and B. Pan). Characteristics of included studies were extracted for stratified analyses and meta-regression. Survival data .Publication bias was assessed by Begg's test and symmetry of funnel plot. Statistical heterogeneity was assessed by Cochrane's Q and I-square statistics. Cochrane's Q test with s et al. . Meta-re


The expression and involvement of estrogen (ER) and progesterone receptor (PR) is extensively studied in endometrial cancer. Androgen receptor (AR) is a hormone receptor less studied in female cancers, and we here aim to investigate the expression level of AR in endometrial cancer precursor lesions, primary tumors and metastases, and its potential as therapeutic target.P < 0.001), and a calculated AR to ERα ratio identified a subgroup of patients with particular poor outcome. The anti-androgen enzalutamide may have a growth inhibitory effect in endometrial cancer cells based on experiments with primary endometrial tumor cells.Expression of AR was observed in 93% of hyperplasias, but only in 41% of non-endometrioid tumors. Compared to estrogen and progesterone receptor AR is more commonly expressed in metastatic lesions, and AR status is discordant in primary and metastatic lesions in a large proportion of cases. AR protein level was significantly associated with survival  were investigated for expression of AR in relation to survival, clinical and histopathological data. Protein levels were investigated by immunohistochemistry and reverse phase protein array; mRNA levels by DNA oligonucleotide microarray. The effect of androgen stimulation and inhibition was tested on primary endometrial tumor cells.A large proportion of metastatic endometrial cancer lesions express AR, which may be a potential target in these patients. Treatment targeting AR may be of particular benefit in patients with high AR levels compared to ERα levels. Endometrial cancer is the most common gynecological malignancy in the Western countries . Little Traditionally, endometrial cancers have been divided into two groups. Type I, accounting for approximately 80% of cases, is associated with endometrioid histology, low stage and grade, good prognosis and estrogen dependency , 4. TypeAndrogen receptor (AR) is a hormone receptor less studied in endometrial cancer, although a target for treatment in other cancers , 10. It Given the similarities of breast and endometrial cancers, exploring AR expression in endometrial cancer might reveal new therapeutic strategies. Additionally, AR status in malignant endometrial lesions may be related to clinical phenotype and could represent a novel biomarker for prognosis.The expression pattern of AR was investigated in relation to aggressiveness of endometrial cancer. AR staining was predominantly nuclear, and only staining in glandular tissue was scored Figure  and 1B. As for most cancers, metastatic endometrial cancers have high mortality and few treatment options. Characterization of metastatic lesions is therefore highly important, especially considering molecular targets with existing therapeutics. Expression was investigated in 298 metastatic lesions from 142 patients . We obseP < 0.001), non-endometrioid histology (P < 0.001) and high grade within the endometrioid subgroup (P = 0.001) , indicating that loss of AR may not add additional information regarding survival when used in a clinical setting. Still, the high number of primary tumors and metastatic lesions with intact expression of AR could point to an unexploited potential for treatment targeting AR in endometrial cancer, and it might be of particular interest in specific subgroups as observed for other cancer types.Loss of AR was significantly associated with established features of aggressive tumors, such as high FIGO stage P < 0.00, non-end1) Table  and with1) Table . In multBased on previous findings in breast cancer, we hypothesized that also for endometrial cancer, the effect of AR signaling may be influenced by the presence of ERα. Interestingly, the patients with the highest calculated AR to ERα ratio (based on RPPA data) had significantly worse survival, both in the whole population Figure  and in FThe underlying mechanisms involved were explored by investigating transcriptional alterations related to the high AR to ERα ratio group. In GSEA analysis several of the top ranked GO gene sets enriched in the high AR to ERα ratio group were associated with cell cycle regulation . This fiTo explore if transcriptional alterations related to the high AR to ERα ratio could suggest new targets for treatment, Connectivity map was queried for drug signatures negatively correlated with the gene expression profile of tumors with a high AR to ERα ratio. Compounds targeting phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway were among the top ranked, along with HSP90 inhibitors, known to disrupt hormone binding and hormone receptor stability , the AR Several studies have demonstrated that ERα and PR status are important prognostic biomarkers, also predicting response to anti-hormonal therapy in endometrial cancer , 7. For in vitro [et. al. found that a high AR to ERα ratio was an independent predictor of disease-free and disease specific survival in breast cancer [ClinicalTrials.gov Identifier NCT00468715, NCT01889238). Similar studies in endometrial cancer patients could reveal whether AR could be a potential target also in endometrial cancer patients with high AR to ERα ratio.The extensive experience with anti-androgen treatment in prostate cancer makes it attractive for investigation also in other hormone dependent cancers. There are several available anti-androgen treatments with different mechanism of action. The anti-androgen bicalutamide inhibits binding of ligand to the androgen receptor, while enzalutamide, a second generation anti-androgen, inhibits several steps of AR activation. Inhibition of AR signaling can also be achieved through inhibition of ligand synthesis. Abiraterone inhibits the enzyme CYP17 which is necessary for androgen biosynthesis . Althougin vitro , 22, andin vitro , 24. Thein vitro , 33. Coct cancer . StudiesHormonal treatment targeting ERα and PR is found to be most effective if the hormone receptor is expressed , 35. SinA population-based patient series was prospectively collected from 2001 to 2015 and included 718 primary tumors from patients diagnosed with endometrial cancer in Hordaland County (Norway). Clinical data were collected as previously described  and patiThe study has been approved by Norwegian legislation, including the Norwegian Data Inspectorate, Norwegian Social Sciences Data Services and the Western Regional Committee for Medical and Health Research Ethics (REK 2009/2315). Patients gave written informed consent.Tissue microarrays (TMA) were dewaxed in xylene and rehydrated in graded ethanol series before microwave boiling in target retrieval buffer (pH9) for 15 minutes. Staining for AR, ERα and PR expression was performed as previously described , 7, 40. RNA was extracted from fresh frozen tissue from 18 hyperplasias, 174 primary tumors and 42 metastases, and hybridized to Agilent Whole Human Genome Microarrays 44k (Cat. No. G4112F), according to the manufacturer's instruction. The arrays were scanned and normalized as previously described . Gene Onhttps://www.mdanderson.org/education-and-research/resources-for-professionals/scientific-resources/core-facilities-and-services/functional-proteomics-rppa-core/index.html). Relative protein levels were determined by fitting each dilution curve with a logistic model (‘Supercurve Fitting’ http://bioinformatics.mdanderson.org/OOMPA) [Reverse Phase Protein Array (RPPA) was performed for 370 of the primary tumors as previously described , 46. Slig/OOMPA) . ExpressData were analyzed using SPSS version 22 . Probability < 0.05 was considered statistical significant. Associations between groups were evaluated using the chi-square test for categorical variables and the Mann-Whitney U test for continuous variables. Univariate survival analysis was performed using the Kaplan Meier (product-limit) method. Date of primary surgery was entry date, and date of death due to endometrial carcinoma was defined as event for estimation of disease specific survival. Survival between groups was compared using the log-rank (Mantel-Cox) test. The Cox proportional hazard regression model was used to evaluate the prognostic impact of AR adjusted for other prognostic parameters. Included in the model was in addition to AR; age, histologic type and grade, all factors with known prognostic impact in endometrial cancer.