Inhibiting miR-618 Promotes Keratinocytes Proliferation and Migration to Enhance Wound Healing in Mice.

The delay in wound healing caused by chronic wounds or pathological scars is a pressing issue in clinical practice, imposing significant economic and psychological burdens on patients. In particular, with the aging of the population and the increasing incidence of diseases such as diabetes, impaired wound healing is one of the growing health problems. MicroRNA (miRNA) plays a crucial role in wound healing and regulates various biological processes. Our results show that miR-618 was significantly upregulated during the inflammatory phase of wound healing.Subsequently, miR-618 promotes the secretion of pro-inflammatory cytokines and regulates the proliferation and migration of keratinocytes. Mechanistically, miR-618 binds to the target gene-Atp11b and inhibits the PI3K-Akt signaling pathway, inhibiting the epithelial-mesenchymal transition (EMT) of keratinocytes. In addition, the PI3K-Akt signaling pathway induces the enrichment of nuclear miR-618, and miR-618 binds to the promoter of Lin7a to regulate gene transcription. Intradermal injection of miR-618 antagomir around full-thickness wounds in peridermal mice effectively accelerates wound closure compared to control. In conclusion, miR-618 antagomir can be a potential therapeutic agent for wound healing.

Comparative histological analysis of cerebellum of representative species of elasmobranchii

SUMMARY: In elasmobranch fishes, variations in gross structural organization of cerebellum has been extensively explored. The basic histological features of cerebellum although conserved in the group but the comparative account on subtle cellular variations is largely underestimated. The present study aims to explore the histological and cellular variations in different layers of cerebellar cortex of the representative elasmobranchs' species belonging to different habitat. Our findings showed that the histological architecture of cerebellar granular layer between the examined species varies noticeably. By and large increase cellular density were observed in all the layers of cerebellum in the representative species of shark compared to ray. The findings were then compared and discussed with reference to their habitat and behavior.

En los peces elasmobranquios, las variaciones en la organización estructural general del cerebelo se han explorado ampliamente. Las características histológicas básicas del cerebelo, aunque se conservan en el grupo, pero la descripción comparativa de las variaciones celulares sutiles es limitada. El presente estudio tiene como objetivo explorar las variaciones histológicas y celulares en diferentes capas de la corteza cerebelosa de las especies representativas de elasmobranquios pertenecientes a diferentes hábitats. Nuestros hallazgos mostraron que la arquitectura histológica de la capa granular del cerebelo entre las especies examinadas varía notablemente. Se observó un gran aumento de la densidad celular en todas las capas del cerebelo en las especies representativas de tiburón en comparación con la raya. Luego, los hallazgos se compararon y discutieron con referencia a su hábitat y comportamiento.

Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G-peptides.

While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand-binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C-terminus of the Gα subunit (G-peptides) on signaling from two Gi-coupled receptors, adenosine A1 receptor (A1 R) and cannabinoid receptor 1 (CB1 ). We expressed A1 R and CB1 fusions with G-peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G-peptides. We used agonists N6 -Cyclopentyladenosine (CPA) and 5'-N-Ethylcarboxamidoadenosine (NECA) for A1 R and 2-Arachidonoylglycerol (2-AG) and WIN 55,212-2 mesylate (WN) for CB1 . G-peptides derived from Gαi and Gαq enhance agonist-dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi-coupled signaling. In contrast, both G-peptides suppress agonist-dependent IP1 levels suggesting that they differentially function as negative allosteric modulators of Gq-coupled signaling. Taken together with our previous studies on Gs-coupled receptors, this study provides an extended model for the allosteric effects of G-peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity.

Minute-scale persistence of a GPCR conformation state triggered by non-cognate G protein interactions primes signaling.

Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor-effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR-G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the ß2-adrenergic receptor (ß2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. ß2-AR-Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.

ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells.

FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the ß2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs-XL, Gαi2, or Gαq subunits. Agonist stimulation of ß2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.

Severe Exacerbation of Myasthenia Gravis Associated with Checkpoint Inhibitor Immunotherapy.

Monoclonal antibodies that target either PD-1 or PD-L1 have recently been approved for treatment of advanced non-small cell lung cancer. These antibodies are immune checkpoint inhibitors which have been shown to exacerbate Myasthenia Gravis (MG) and other autoimmune diseases. While effective in preventing tumor cells from evading immune attack, immune checkpoint inhibitors such as nivolumab, an antibody directed against the programmed cell death protein-1 (PD-1) receptor located on T-cells, may also cause immune dysregulation and could cause or potentiate pre-existing autoimmune conditions. We present a patient with latent ocular MG treated with nivolumab for her stage IV non-small cell lung cancer who developed generalized MG and severe myasthenic crisis. Providers must be aware of the risks inherent to these novel therapies since they can have life-threatening effects.

Priming GPCR signaling through the synergistic effect of two G proteins.

Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, ß2-adrenergic receptor (ß2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (i) sequential G-protein interactions at the cognate site, (ii) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and (iii) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.

Synthesis and in silico study of 2-furyl(4-{4-[(substituted)sulfonyl]benzyl}-1-piperazinyl)methanone derivatives as suitable therapeutic agents

Abstract In the study presented here, a new series of 2-furyl(4-{4-[(substituted)sulfonyl]benzyl}-1-piperazinyl)methanone derivatives was targeted. The synthesis was initiated by the treatment of different secondary amines (1a-h) with 4-bromomethylbenzenesulfonyl chloride (2) to obtain various 1-{[4-(bromomethyl)phenyl]sulfonyl}amines (3a-h). 2-Furyl(1-piperazinyl)methanone (2-furoyl-1-piperazine; 4) was then dissolved in acetonitrile, with the addition of K2CO3, and the mixture was refluxed for activation. This activated molecule was further treated with equi-molar amounts of 3a-h to form targeted 2-furyl(4-{4-[(substituted)sulfonyl]benzyl}-1-piperazinyl)methanone derivatives (5a-h) in the same reaction set up. The structure confirmation of all the synthesized compounds was carried out by EI-MS, IR and 1H-NMR spectral analysis. The compounds showed good enzyme inhibitory activity. Compound 5h showed excellent inhibitory effect against acetyl- and butyrylcholinesterase with respective IC50 values of 2.91±0.001 and 4.35±0.004 µM, compared to eserine, a reference standard with IC50 values of 0.04±0.0001 and 0.85±0.001 µM, respectively, against these enzymes. All synthesized molecules were active against almost all Gram-positive and Gram-negative bacterial strains tested. The cytotoxicity of the molecules was also checked to determine their utility as possible therapeutic agents.

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay.

FÓ§rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between Gα subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the ß2-adrenergic receptor and the Gαs C-terminus peptide upon treatment with 100 µM isoproterenol hydrochloride as previously characterized(1). Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a Gα C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the ß2-adrenergic receptor (ß2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in ß2-AR and V1AR, respectively. The ß2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with ß2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.

Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.

Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved (E/D)RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D)RY motif (ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the ß2-AR (E/D)RY ionic lock enhances interaction with Gs. However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D)RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of ß2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with Gs and promotes Gi binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.

Pakistani urban population demonstrates a poor knowledge about cancers: a pilot survey.

BACKGROUND: This cross-sectional study was carried out to assess the perceptions and level of knowledge of a Pakistani urban population regarding presentations, risk factors, and screening of cancers. METHODS: From 4 tertiary care hospitals, 439 subjects were recruited through convenient sampling method and interviewed using a standard questionnaire. RESULTS: More than 90% of subjects demonstrated poor knowledge. Good knowledge was associated with female gender, being married, higher socioeconomic status, and level of education. CONCLUSIONS: Our sample demonstrates an inadequate knowledge regarding the presentations, risk factors, and screening of cancers. Efforts aiming to correct these deficiencies might result in a successful and cost-effective model for primary and secondary prevention of major cancers in Pakistan.

Socio-demographic correlates of betel, areca and smokeless tobacco use as a high risk behavior for head and neck cancers in a squatter settlement of Karachi, Pakistan.

BACKGROUND: Head and neck cancers are a major cancer burden in Pakistan. They share a common risk factor profile including regular consumption of products of betel, areca and tobacco. Use of paan, chaalia, gutka, niswar and tumbaku is acceptable in Pakistan and is considered a normal cultural practice. This cross-sectional study was carried out to understand the relation of socio-demographic factors for the consumption of paan, chaalia, gutka, niswar and tumbaku in Pakistani population. Through systematic sampling, 425 subjects from a squatter settlement in Karachi were interviewed using a structured questionnaire. High risk behavior was defined as Daily use of any of the above products. RESULTS: Daily use of all the substances except chaalia was higher among males compared to females. Chaalia use was higher among adolescents than adults while non-married consumed both chaalia and gutka more than married. Mohajir ethnicity had higher prevalence of paan, gutka and tumbaku use while Pathans had higher prevalence of niswar use. CONCLUSION: Prevalence of use of chewable products is high in Pakistan with particularly high use of certain substances related with socio-demographic profiles. Industrially prepared products, chaalia and gutka, are gaining popularity among youth. Policies and focused interventions can be developed taking into consideration the preferred use of products among different socio-demographic groups.

Chewing of betel, areca and tobacco: perceptions and knowledge regarding their role in head and neck cancers in an urban squatter settlement in Pakistan.

The link of betel, areca and chewable tobacco with head and neck cancers is clearly established. Fifty eight percent of the global head and neck cancers occur in South and Southeast Asia, where chewing of betel, areca and tobacco are common. This study was carried out to establish the pattern of use of Paan, Chaalia, Gutka, Niswar, Tumbaku and Naas among population of squatter settlement of Karachi and to determine the perceptions and knowledge regarding their role in the etiology of head and neck cancers. It was a cross-sectional study, performed at Bilal colony in Karachi. Through systematic sampling, 425 subjects [a male and female from a household] were interviewed with a structured questionnaire. Knowledge regarding etiology of head and neck cancers was classified in ordinals of "good", "some" and "poor", for each substance separately, while practices were classified into "daily user", "occasional user" and "never user". About 40% of the participants were chewing at least one item [betel, areca or tobacco products] on daily basis. This prevalence was 2.46 times higher among males than females and 1.39 times higher among adolescents than adults. At least 79% of the participants were classified as having poor knowledge about the carcinogenicity of each of these items. Knowledge increased with age and level of education. Health hazards of these items were poorly recognized and about 20% perceived at least one of these items to be beneficial. Positive attitudes were seen regarding the steps to curb the production, business and consumption of these substances. In conclusion, prevalence of chewing of betel, areca and tobacco among adults and adolescents is high. Deficiency in knowledge and wrong perception of favorable effect of chewing products is common. Besides curtailing the availability of chewing products, correct knowledge regarding its ill-effects should be inculcated among population to decrease the burden of head and neck cancers.