Tas2R activation relaxes airway smooth muscle by release of Gαt targeting on AChR signaling.

Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD.

Repositioning baloxavir marboxil as VISTA agonist that ameliorates experimental asthma.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), a novel negative checkpoint regulator, plays an essential role in allergic pulmonary inflammation in mice. Treatment with a VISTA agonistic antibody could significantly improve asthma symptoms. Thus, for allergic asthma treatment, VISTA targeting may be a compelling approach. In this study, we examined the functional mechanism of VISTA in allergic pulmonary inflammation and screened the FDA-approved drugs for VISTA agonists. By using mass cytometry (CyTOF), we found that VISTA deficiency primarily increased lung macrophage infiltration in the OVA-induced asthma model, accompanied by an increased proportion of M1 macrophages (CD11b+F4/80+CD86+) and a decreased proportion of M2 macrophages (CD11b+F4/80+CD206+). Further in vitro studies showed that VISTA deficiency promoted M1 polarization and inhibited M2 polarization of bone marrow-derived macrophages (BMDMs). Importantly, we discovered baloxavir marboxil (BXM) as a VISTA agonist by virtual screening of FDA-approved drugs. The surface plasmon resonance (SPR) assays revealed that BXM (KD = 1.07 µM) as well as its active form, baloxavir acid (BXA) (KD = 0.21 µM), could directly bind to VISTA with high affinity. Notably, treatment with BXM significantly ameliorated asthma symptoms, including less lung inflammation, mucus secretion, and the generation of Th2 cytokines (IL-5, IL-13, and IL-4), which were dramatically attenuated by anti-VISTA monoclonal antibody treatment. BXM administration also reduced the pulmonary infiltration of M1 macrophages and raised M2 macrophages. Collectively, our study indicates that VISTA regulates pulmonary inflammation in allergic asthma by regulating macrophage polarization and baloxavir marboxil, and an old drug might be a new treatment for allergic asthma through targeting VISTA.

A Transposon-Associated CRISPR/Cas9 System Specifically Eliminates both Chromosomal and Plasmid-Borne mcr-1 in Escherichia coli.

The global spread of antimicrobial-resistant bacteria has been one of the most severe threats to public health. The emergence of the mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by ISApl1-formed transposon Tn6330 and mediated rapid dispersion among Enterobacteriaceae species. Here, we developed a CRISPR/Cas9 system flanked by ISApl1 in a suicide plasmid capable of exerting sequence-specific curing against the mcr-1-bearing plasmid and killing the strain with chromosome-borne mcr-1. The constructed ISApl1-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne mcr-1 or directly eradicated the bacteria harboring chromosome-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting the mcr-1 gene. This method is highly efficient in removing the mcr-1 gene from Escherichia coli, thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with immunity against the acquisition of the exogenous mcr-1 containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.

ISKpn40-Mediated Mobilization of the Colistin Resistance Gene mcr-3.11 in Escherichia coli.

The mobile colistin resistance gene mcr-3 has globally disseminated since it was first reported in 2017 in Escherichia coliIn vitro mobilization assays in this study demonstrate the functionality of the composite transposon structure ISKpn40-mcr-3.11-dgkA-ISKpn40 in wild-type and recA-E. coli strains. These transpositions generated 4-bp duplications at the target sites. This is the first report demonstrating the mobility of the mcr-3.11 gene by transposition.

Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor-transmembrane protein 16A-voltage-dependent Ca2+ channel axis and contribute to bronchial hyperresponsiveness in asthma.

BACKGROUND: Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness. OBJECTIVE: We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness. METHODS: We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model. RESULTS: Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl- currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity. CONCLUSIONS: A G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.

Regulation of DLK1 by the maternally expressed miR-379/miR-544 cluster may underlie callipyge polar overdominance inheritance.

Inheritance of the callipyge phenotype in sheep is an example of polar overdominance inheritance, an unusual mode of inheritance. To investigate the underlying molecular mechanism, we profiled the expression of the genes located in the Delta-like 1 homolog (Dlk1)-type III iodothyronine deiodinase (Dio3) imprinting region in mice. We found that the transcripts of the microRNA (miR) 379/miR-544 cluster were highly expressed in neonatal muscle and paralleled the expression of the Dlk1. We then determined the in vivo role of the miR-379/miR-544 cluster by establishing a mouse line in which the cluster was ablated. The maternal heterozygotes of young mutant mice displayed a hypertrophic tibialis anterior muscle, extensor digitorum longus muscle, gastrocnemius muscle, and gluteus maximus muscle and elevated expression of the DLK1 protein. Reduced expression of DLK1 was mediated by miR-329, a member of this cluster. Our results suggest that maternal expression of the imprinted miR-379/miR-544 cluster regulates paternal expression of the Dlk1 gene in mice. We therefore propose a miR-based molecular working model for polar overdominance inheritance.

Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.

KEY POINTS: Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+ /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. ABSTRACT: Ca2+ /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1SM+/+ , MYPT1SM-/- and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

Physiological signalling to myosin phosphatase targeting subunit-1 phosphorylation in ileal smooth muscle.

KEY POINTS: The extent of myosin regulatory light chain phosphorylation (RLC) necessary for smooth muscle contraction depends on the respective activities of Ca(2+) /calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP), which contains a regulatory subunit MYPT1 bound to the phosphatase catalytic subunit and myosin. MYPT1 showed significant constitutive T696 and T853 phosphorylation, which is predicted to inhibit MLCP activity in isolated ileal smooth muscle tissues, with additional phosphorylation upon pharmacological treatment with the muscarinic agonist carbachol. Electrical field stimulation (EFS), which releases ACh from nerves, increased force and RLC phosphorylation but not MYPT1 T696 or T853 phosphorylation. The conditional knockout of MYPT1 or the knockin mutation T853A in mice had no effect on the frequency-maximal force responses to EFS in isolated ileal tissues. Physiological RLC phosphorylation and force development in ileal smooth muscle depend on myosin light chain kinase and MLCP activities without changes in constitutive MYPT1 phosphorylation. ABSTRACT: Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+) /calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle-specific deletion of MYPT1; (2) non-phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild-type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild-type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation.

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction.

KEY POINTS: Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. ABSTRACT: Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.

Myosin phosphatase target subunit 1 (MYPT1) regulates the contraction and relaxation of vascular smooth muscle and maintains blood pressure.

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.

Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.

Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

Altered contractile phenotypes of intestinal smooth muscle in mice deficient in myosin phosphatase target subunit 1.

BACKGROUND & AIMS: The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca(2+)-dependent myosin regulatory light chain. We generated mice with a smooth muscle-specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction. METHODS: We used the Cre-loxP system to establish Mypt1-floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle-specific deletion of MYPT1 (Mypt1(SMKO) mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses. RESULTS: Young adult Mypt1(SMKO) mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1(SMKO) mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1(SMKO) mice had increased sensitivity and contraction in response to Ca(2+). CONCLUSIONS: MYPT1 is not essential for smooth muscle function in mice but regulates the Ca(2+) sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.

Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria.

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.

[Clinical and pathological features in children with progressive muscular dystrophy].

OBJECTIVE: To investigate the clinical and pathological features of progressive muscular dystrophy (PMD) in children and to provide help for the early and accurate diagnosis of PMD. METHODS: Retrospective analysis was performed on the clinical data of 99 hospitalized children with PMD, including clinical manifestations, age of onset, family history, creatase, electromyogram (EMG) and pathological changes of muscles. RESULTS: Of the 99 children with PMD, the age of onset was 0.5-14.5 (4.7 ± 3.1) years. Eleven cases (11%) had a family history of PMD. Twenty-six (26%) were misdiagnosed as other diseases. All patients presented with muscle weakness when seeing the doctor, and 66 (67%) of them had muscle atrophy and/or hypertrophy. All patients had elevated creatine kinase (CK) levels. The 2-7-year-old group (n=51) had a mean CK level of 9965 ± 8876 U/L, and the 7-15-year-old group (n=48) had a mean CK level of 5110 ± 4498 U/L, with a significant difference between the two groups (P<0.01). The EMG examination performed on 66 patients showed that 54 cases (82%) had myogenic damage and 10 cases (15%) had neurogenic damage. Light microscopy revealed coexistence of atrophy and hypertrophy of muscle fibers, hyaline degeneration and granular degeneration. Electron microscopy showed that muscle fibers were different in thickness, some atrophic or hypertrophic; muscle cell nuclei moved inwardly, myofilaments dissolved and disappeared mildly under the sarcolemma, there were scattered melting lesions within muscle fibers, the numbers of glycogen granules and mitochondria increased, mild hyperplasia and expansion of sarcoplasmic reticulum were seen, and a small number of muscle fibers had necrosis. CONCLUSIONS: Weakness of both lower extremities remains the main reason for PMD patients seeing the doctor. CK is the main laboratory indicator for diagnosis of PMD. PMD is mainly manifested as myogenic damage in the early stage and may be accompanied by neurogenic damage in the late stage, according to the EMG examination. With a high misdiagnosis rate, PMD may be misdiagnosed as many other diseases. Pathological examination under light microscope and electron microscope is the main means for confirming a PMD diagnosis.

Gonadotropin-releasing hormone receptor inhibits triple-negative breast cancer proliferation and metastasis.

BACKGROUND: Gonadotropin-releasing hormone receptor (GnRHR) is expressed in several malignant tumors and inhibits the proliferation and metastasis of cancer cells, but its role in triple-negative breast cancers (TNBCs) is unclear. This study investigated the biological effects of GnRHR and their influence on TNBC prognosis. METHODS: The GSE21653 database was used to obtain information about GnRHR expression and clinicopathological factors in patients with TNBC. GnRHR was activated in cultured MDA-MB-231 and MDA-MB-468 cells by leuprolide acetate and antagonized by elagolix sodium. Cell proliferation was assessed by the cell counting kit-8 and colony formation assays. Cell metastasis was detected by the wound healing assay and Transwell assay. Apoptosis and the cell cycle were investigated by flow cytometry. GnRHR protein expression was determined by western blotting. RESULTS: GnRHR mRNA expression was significantly higher in patients with TNBC than in hormone receptor+/human epidermal growth factor receptor (HER)2- and HER2+ patients with breast cancer. Patients with high GnRHR expression had significantly better disease-free survival than those with lower expression. Activated GnRHR significantly inhibited cell proliferation and metastasis, increased apoptosis, and enhanced GnRHR protein expression levels. CONCLUSION: GnRHR inhibits TNBC proliferation and metastasis, suggesting it could be targeted for TNBC treatment.

Case report: Blepharospasm in peak-dose dyskinesia may benefit from amantadine in Parkinson's disease.

Introduction: Blepharospasm is uncommon in Parkinson's disease, especially in the peak-dose dyskinesia period. Case presentation: We herein present the case of a patient with PD who developed blepharospasm in the peak-dose dyskinesia period. The symptom was improved by taking amantadine. Conclusion: The current report expands the phenomenology of peak-dose dykinesia in PD to include dystonic blepharospasm. This complication of levodopa therapy may respond to amantadine despite the dystonic appearance of movements.

MYPT1 reduction is a pathogenic factor of erectile dysfunction.

Erectile dysfunction (ED) is closely associated with smooth muscle dysfunction, but its underlying mechanisms remains incompletely understood. We here reported that the reduced expression of myosin phosphatase target subunit 1 (MYPT1), the main regulatory unit of myosin light chain phosphatase, was critical for the development of vasculogenic ED. Male MYPT1 knockout mice had reduced fertility and the penises displayed impaired erections as evidenced by reduced intracavernous pressure (ICP). The penile smooth muscles of the knockout mice displayed enhanced response to G-Protein Couple Receptor agonism and depolarization contractility and resistant relaxation. We further identified a natural compound lotusine that increased the MYPT1 expression by inhibiting SIAH1/2 E3 ligases-mediated protein degradation. This compound sufficiently restored the ICP and improved histological characters of the penile artery of Mypt1 haploinsufficiency mice. In diabetic ED mice (db/db), the decreased expression of MYPT1 was measured, and ICP was improved by lotusine treatment. We conclude that the reduction of MYPT1 is the major pathogenic factor of vasculogenic ED. The restoration of MYPT1 by lotusine improved the function of injured penile smooth muscles, and could be a novel strategy for ED therapy.

[A comparative study on psychological and behavioral development in children aged 1 to 3 years fostered by grandparents and by parents].

OBJECTIVE: To investigate the differences of psychological and behavioral development between children aged 1 to 3 years fostered by grandparents and those by parents. METHODS: Psychological and behavioral development of 443 children aged 1 to 3 years fostered by their grandparents and of aged-matched 443 children fostered by their parents were assessed with DST, an intellectual developmental screening test developed by Pediatric Hospital of Fudan University in Shanghai. RESULTS: The abilities of social adaptation and intelligence development in children fostered by their grandparents were obviously retarded as compared with those in children fostered by their parents. CONCLUSIONS: There are shortcomings in psychological and behavioral development in children aged 1 to 3 years fostered by grandparents.

Synergistic antitumor activity of artesunate and HDAC inhibitors through elevating heme synthesis via synergistic upregulation of ALAS1 expression.

Artemisinin and its derivatives (ARTs) were reported to display heme-dependent antitumor activity. On the other hand, histone deacetylase inhibitors (HDACi) were known to be able to promote heme synthesis in erythroid cells. Nevertheless, the effect of HDACi on heme homeostasis in non-erythrocytes remains unknown. We envisioned that the combination of HDACi and artesunate (ARS) might have synergistic antitumor activity through modulating heme synthesis. In vitro studies revealed that combination of ARS and HDACi exerted synergistic tumor inhibition by inducing cell death. Moreover, this combination exhibited more effective antitumor activity than either ARS or HDACi monotherapy in xenograft models without apparent toxicity. Importantly, mechanistic studies revealed that HDACi coordinated with ARS to increase 5-aminolevulinate synthase (ALAS1) expression, and subsequent heme production, leading to enhanced cytotoxicity of ARS. Notably, knocking down ALAS1 significantly blunted the synergistic effect of ARS and HDACi on tumor inhibition, indicating a critical role of ALAS1 upregulation in mediating ARS cytotoxicity. Collectively, our study revealed the mechanism of synergistic antitumor action of ARS and HDACi. This finding indicates that modulation of heme synthesis pathway by the combination based on ARTs and other heme synthesis modulators represents a promising therapeutic approach to solid tumors.

THAP7 promotes cell proliferation by regulating the G1/S phase transition via epigenetically silencing p21 in lung adenocarcinoma.

PURPOSE: Lung adenocarcinoma (LUAD) is one of the most common cancers worldwide. The THanatos-Associated Proteins (THAP) family plays an essential role in multiple cancers. However, the role of THAP7 in cancers has remained elusive. METHODS: THAP7 expression status in LUAD tissues was analysed by using the Oncomine database and qRT-PCR, and its expression level in LUAD cell lines was detected by qRT-PCR and Western blotting. The role of THAP7 in LUAD cells was determined by proliferation, colony formation, and cell cycle analyses. In vivo role of THAP7 was studied on xenograft models. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to determine the activity and acetylation of the p21 promoter. RESULTS: THAP7 expression was increased in LUAD tissues and cell lines. Moreover, the high expression of THAP7 was correlated with poor prognosis. The overexpression of THAP7 accelerated the G1/S phase transition and promoted tumour growth both in vitro and in vivo. A mechanistic study revealed that THAP7 reduced the acetylation of histone H3 on the p21 promoter to suppress p21 transcription. CONCLUSION: For the first time, we demonstrated the function of THAP7 in LUAD, and our findings suggested that THAP7 may be a potential molecular therapy target in LUAD.