Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.

Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.

Direct Observation of Z-Scheme Route in Cu31 S16 /Znx Cd1-x S Heteronanoplates for Highly Efficient Photocatalytic Hydrogen Evolution.

Although photocatalytic hydrogen production from water holds great potential as a renewable and sustainable energy alternative, the practical application of the technology demands cost-effective, simple photocatalytic systems with high efficiency in hydrogen evolution reaction (HER). Herein, the synthesis and characterization of Cu31 S16 /Znx Cd1-x S heterostructured nanoplates (Cu31 S16 /ZnCdS HNPs) as a high photocatalytic system are reported. The cost-effective, hierarchical structures are easily prepared using the Cu31 S16 NPs as the seed by the epitaxial growth of the ZnCdS nanocrystals (NCs). The Cu31 S16 /ZnCdS without the noble metal cocatalyst exhibits a high HER rate of 61.7 mmol g-1  h-1 , which is 8,014 and 17 times higher than that of Cu31 S16 and ZnCdS, respectively, under visible light irradiation. The apparent quantum yield (AQY) of Cu31 S16 /ZnCdS reaches 67.9% at 400 nm with the highest value so far in the reported ZnCdS-based photocatalysts. The excellent activity and stability of the Cu31 S16 /ZnCdS are attributed to the formation of a strong internal electric field (IEF) and the Z-scheme pathway. The comprehensive experiments and theoretical calculations provide the direct evidences of the Z-scheme route. This work may offer a way for the design and development of efficient photocatalysts to achieve solar-to-chemical energy conversion at a practically useful level.

Modulation of DOM-Induced Head-Twitch Response by mGluR2 Agonist/Inverse Agonist is Associated with 5-HT2AR-Mediated Gs Signaling Pathway.

Hallucinogenic 5-HT2A receptor (5-HT2AR) agonists-induced head-twitch response (HTR) is regulated by Gs signaling pathway. Formation of heterodimers between 5-HT2AR and metabotropic glutamate mGlu2 receptor (mGluR2) is essential for the hallucinogenic 5-HT2AR agonist-induced HTR. In order to investigate the effects of mGluR2 agonists and inverse agonists on hallucinogenic 5-HT2AR agonists DOM-induced HTR, C57BL/6 mice were pretreated with mGluR2 agonists (LY379268, LY354740, LY404039) or the inverse agonist LY341495, and the HTR was manually counted after administering DOM immediately. IP-One (IP1) HTRF assay and cAMP assay were performed to evaluate the effect of LY341495 or LY354740 on DOM-induced Gq and Gs activation in Human Embryonic Kidney-293 (HEK-293) T-type cells co-expressing 5-HT2AR and mGluR2. The results showed that DOM-induced HTR in mice was dose-dependently inhibited by LY379268, LY354740, and LY404039, while it was dose-dependently enhanced by LY341495. Moreover, LY341495 reversed the inhibitory effect of LY354740 on DOM-induced HTR. In HEK-293T cells co-expressing 5-HT2AR and mGluR2, DOM-induced cAMP level was decreased by LY354740 and increased by LY341495, but DOM-induced IP1 level was not regulated by LY354740 or LY341495. The regulation of DOM-induced HTR by mGluR2 agonists and inverse agonists is closely related to 5-HT2AR-mediated Gs signaling pathway. In HEK-293T cells co-expressing 5-HT2AR and mGluR2 A677S/A681P/A685G mutant (mGluR2 3 A mutant), DOM-induced cAMP level was not regulated by LY354740, but was significantly enhanced by LY341495. The 5-HT2AR/mGluR2 heterodimers is critical for DOM-induced HTR and cAMP level, both of which are inhibited by mGluR2 agonists and enhanced by mGluR2 inverse agonists.

Interhelical E@g-N@a interactions modulate coiled coil stability within a de novo set of orthogonal peptide heterodimers.

The designability of orthogonal coiled coil (CC) dimers, which draw on well-established design rules, plays a pivotal role in fueling the development of CCs as synthetically versatile assembly-directing motifs for the fabrication of bionanomaterials. Here, we aim to expand the synthetic CC toolkit through establishing a "minimalistic" set of orthogonal, de novo CC peptides that comprise 3.5 heptads in length and a single buried Asn to prescribe dimer formation. The designed sequences display excellent partner fidelity, confirmed via circular dichroism (CD) spectroscopy and Ni-NTA binding assays, and are corroborated in silico using molecular dynamics (MD) simulation. Detailed analysis of the MD conformational data highlights the importance of interhelical E@g-N@a interactions in coordinating an extensive 6-residue hydrogen bonding network that "locks" the interchain Asn-Asn' contact in place. The enhanced stability imparted to the Asn-Asn' bond elicits an increase in thermal stability of CCs up to ~15°C and accounts for significant differences in stability within the collection of similarly designed orthogonal CC pairs. The presented work underlines the utility of MD simulation as a tool for constructing de novo, orthogonal CCs, and presents an alternative handle for modulating the stability of orthogonal CCs via tuning the number of interhelical E@g-N@a contacts. Expansion of CC design rules is a key ingredient for guiding the design and assembly of more complex, intricate CC-based architectures for tackling a variety of challenges within the fields of nanomedicine and bionanotechnology.

BMP heterodimers signal via distinct type I receptor class functions.

Heterodimeric TGF-ß ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal-ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers. Instead, we find that while overexpressed BMP2 homodimers can signal, they require two nonredundant type I receptors, one from the Acvr1 subfamily and one from the Bmpr1 subfamily. This implies that all BMP signaling within the zebrafish gastrula, even BMP2 homodimer signaling, requires Acvr1. This is particularly surprising as BMP2 homodimers do not bind Acvr1 in vitro. Furthermore, we find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the potency of the Bmp2/7 heterodimer arises from the ability to recruit both Acvr1 and Bmpr1 into the same signaling complex.

Design, synthesis, and bioactivity evaluation of clindamycin derivatives against multidrug-resistant bacterial strains.

Our research aims to reduce the bacterial resistance of clindamycin against Gram-positive bacteria and expand its range of bacterial susceptibility. First, we optimized the structure of clindamycin based on its structure-activity relationship. Second, we employed the fractional inhibitory concentration method to detect drugs suitable for combination with clindamycin derivatives. We then used a linker to connect the clindamycin derivatives with the identified combined therapy drugs. Finally, we tested antibacterial susceptibility testing and conducted in vitro bacterial inhibition activity assays to determine the compounds. with the highest efficacy. The results of our study show that we synthesized clindamycin propionate derivatives and clindamycin homo/heterodimer derivatives, which exhibited superior antibacterial activity compared to clindamycin and other antibiotics against both bacteria and fungi. In vitro bacteriostatic activity testing against four types of Gram-negative bacteria and one type of fungi revealed that all synthesized compounds had bacteriostatic effects at least 1000 times better than clindamycin and sulfonamides. The minimum inhibitory concentration (MIC) values for these compounds ranged from 0.25 to 0.0325 mM. Significantly, compound 5a demonstrated the most potent inhibitory activity against three distinct bacterial strains, displaying MIC values spanning from 0.0625 to 0.0325 mM. Furthermore, our calculations indicate that compound 5a is safe for cellular use. In conclusion, the synthesized compounds hold great promise in addressing bacterial antibiotic resistance.

PDGFRß regulates craniofacial development through homodimers and functional heterodimers with PDGFRα.

Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. While platelet-derived growth factor (PDGF) receptor α (PDGFRα) has well-documented functions in this process, the role of PDGFRß in murine craniofacial development is not well established. We demonstrate that PDGFRα and PDGFRß are coexpressed in the craniofacial mesenchyme of mid-gestation mouse embryos and that ablation of Pdgfrb in the neural crest lineage results in increased nasal septum width, delayed palatal shelf development, and subepidermal blebbing. Furthermore, we show that the two receptors genetically interact in this lineage, as double-homozygous mutant embryos exhibit an overt facial clefting phenotype more severe than that observed in either single-mutant embryo. We reveal a physical interaction between PDGFRα and PDGFRß in the craniofacial mesenchyme and demonstrate that the receptors form functional heterodimers with distinct signaling properties. Our studies thus uncover a novel mode of signaling for the PDGF family during vertebrate development.

G Protein-Coupled Receptor Dimerization-What Next?

Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.

Targeting CRAF kinase in anti-cancer therapy: progress and opportunities.

The RAS/mitogen-activated protein kinase (MAPK) signaling cascade is commonly dysregulated in human malignancies by processes driven by RAS or RAF oncogenes. Among the members of the RAF kinase family, CRAF plays an important role in the RAS-MAPK signaling pathway, as well as in the progression of cancer. Recent research has provided evidence implicating the role of CRAF in the physiological regulation and the resistance to BRAF inhibitors through MAPK-dependent and MAPK-independent mechanisms. Nevertheless, the effectiveness of solely targeting CRAF kinase activity remains controversial. Moreover, the kinase-independent function of CRAF may be essential for lung cancers with KRAS mutations. It is imperative to develop strategies to enhance efficacy and minimize toxicity in tumors driven by RAS or RAF oncogenes. The review investigates CRAF alterations observed in cancers and unravels the distinct roles of CRAF in cancers propelled by diverse oncogenes. This review also seeks to summarize CRAF-interacting proteins and delineate CRAF's regulation across various cancer hallmarks. Additionally, we discuss recent advances in pan-RAF inhibitors and their combination with other therapeutic approaches to improve treatment outcomes and minimize adverse effects in patients with RAF/RAS-mutant tumors. By providing a comprehensive understanding of the multifaceted role of CRAF in cancers and highlighting the latest developments in RAF inhibitor therapies, we endeavor to identify synergistic targets and elucidate resistance pathways, setting the stage for more robust and safer combination strategies for cancer treatment.

Stable Dication Diradicals of Triply Fused Metallo Chlorin-Porphyrin Heterodimers: Impact of the Bridge on the Control of Spin Coupling to Reactivity.

We report an unexpected rearrangement, controlled by the nature of the bridge, leading to the formation of novel, remarkably stable triply fused dinickel(II)/dicopper(II) chlorin-porphyrin dication diradical heterodimers in excellent yields. Here, a dipyrromethene bridge gets completely fused between two porphyrin macrocycles with two new C-C and one C-N bonds. The two macrocycles exhibit extensive π-conjugation through the bridge, which results in an antiferromagnetic coupling between the two π-cation radicals. In addition, the macrocyclic distortion also favours a rare intramolecular ferromagnetic interaction between the CuII and π-cation radical spins to form a triplet state. The structural and electronic perturbation in the unconjugated dication diradical possibly enables the bridging pyrrolic nitrogen to undergo a nucleophilic attack at the nearby ß-carbon of the porphyrin π-cation radical with a computed free energy barrier of >20 kcal mol-1 which was supplied in the form of reflux condition to initiate such a rearrangement process. UV-vis, EPR and ESI-MS spectroscopies were used to monitor the rearrangement process in situ in order to identify the key reactive intermediates leading to such an unusual transformation.

Quinpirole ameliorates nigral dopaminergic neuron damage in Parkinson's disease mouse model through activating GHS-R1a/D2R heterodimers.

Growth hormone secretagogue receptor 1a (GHS-R1a) is an important G protein-coupled receptor (GPCR) that regulates a variety of functions by binding to ghrelin. It has been shown that the dimerization of GHS-R1a with other receptors also affects ingestion, energy metabolism, learning and memory. Dopamine type 2 receptor (D2R) is a GPCR mainly distributed in the ventral tegmental area (VTA), substantia nigra (SN), striatum and other brain regions. In this study we investigated the existence and function of GHS-R1a/D2R heterodimers in nigral dopaminergic neurons in Parkinson's disease (PD) models in vitro and in vivo. By conducting immunofluorescence staining, FRET and BRET analyses, we confirmed that GHS-R1a and D2R could form heterodimers in PC-12 cells and in the nigral dopaminergic neurons of wild-type mice. This process was inhibited by MPP+ or MPTP treatment. Application of QNP (10 µM) alone significantly increased the viability of MPP+-treated PC-12 cells, and administration of quinpirole (QNP, 1 mg/kg, i.p. once before and twice after MPTP injection) significantly alleviated motor deficits in MPTP-induced PD mice model; the beneficial effects of QNP were abolished by GHS-R1a knockdown. We revealed that the GHS-R1a/D2R heterodimers could increase the protein levels of tyrosine hydroxylase in the SN of MPTP-induced PD mice model through the cAMP response element binding protein (CREB) signaling pathway, ultimately promoting dopamine synthesis and release. These results demonstrate a protective role for GHS-R1a/D2R heterodimers in dopaminergic neurons, providing evidence for the involvement of GHS-R1a in PD pathogenesis independent of ghrelin.

Design and synthesis of guaianolide-germacranolide heterodimers as novel anticancer agents against hepatocellular carcinoma.

Inspired by our previous finding that disesquiterpenoids showed more potent antihepatoma cytotoxicity than their corresponding parent monomers, natural product-like guaianolide-germacranolide heterodimers were designed and synthesized from guaianolide diene and germacranolides via a biomimetic Diels-Alder reaction to provide three antihepatoma active dimers with novel scaffolds. To explore the structure-activity relationship, 31 derivatives containing ester, carbamate, ether, urea, amide, and triazole functional groups at C-14' were synthesized and evaluated for their cytotoxic activities against HepG2, Huh7, and SK-Hep-1 cell lines. Among them, 25 compounds were more potent than sorafenib against HepG2 cells, 15 compounds were stronger than sorafenib against Huh7 cells, and 17 compounds were stronger than sorafenib against SK-Hep-1 cells. Compound 23 showed the most potent cytotoxicity against three hepatoma cell lines with IC50 values of 4.4 µM (HepG2), 3.7 µM (Huh7), and 3.1 µM (SK-Hep-1), which were 2.7-, 2.2-, and 2.8-fold more potent than sorafenib, respectively. The underlying mechanism study demonstrated that compound 23 could induce cell apoptosis, prevent cell migration and invasion, cause G2/M phase arrest in SK-Hep-1 cells. Network pharmacology analyses predicted PDGFRA was one of the potential targets of compound 23, and surface plasmon resonance (SPR) assay verified that 23 had strong affinity with PDGFRA with a dissociatin constant (KD) value of 90.2 nM. These promising findings revealed that structurally novel guaianolide-germacranolide heterodimers might provide a new inspiration for the discovery of antihepatoma agents.

Heteromerization of Dopamine D2 and Oxytocin Receptor in Adult Striatal Astrocytes.

The ability of oxytocin (OT) to interact with the dopaminergic system through facilitatory D2-OT receptor (OTR) receptor-receptor interaction in the limbic system is increasingly considered to play roles in social or emotional behavior, and suggested to serve as a potential therapeutic target. Although roles of astrocytes in the modulatory effects of OT and dopamine in the central nervous system are well recognized, the possibility of D2-OTR receptor-receptor interaction in astrocytes has been neglected. In purified astrocyte processes from adult rat striatum, we assessed OTR and dopamine D2 receptor expression by confocal analysis. The effects of activation of these receptors were evaluated in the processes through a neurochemical study of glutamate release evoked by 4-aminopyridine; D2-OTR heteromerization was assessed by co-immunoprecipitation and proximity ligation assay (PLA). The structure of the possible D2-OTR heterodimer was estimated by a bioinformatic approach. We found that both D2 and OTR were expressed on the same astrocyte processes and controlled the release of glutamate, showing a facilitatory receptor-receptor interaction in the D2-OTR heteromers. Biochemical and biophysical evidence confirmed D2-OTR heterodimers on striatal astrocytes. The residues in the transmembrane domains four and five of both receptors are predicted to be mainly involved in the heteromerization. In conclusion, roles for astrocytic D2-OTR in the control of glutamatergic synapse functioning through modulation of astrocytic glutamate release should be taken into consideration when considering interactions between oxytocinergic and dopaminergic systems in striatum.

Alkylbenzoic and Alkyloxybenzoic Acid Blending for Expanding the Liquid Crystalline State and Improving Its Rheology.

Thermotropic mesogens typically exist as liquid crystals (LCs) in a narrow region of high temperatures, making lowering their melting point with the temperature expansion of the mesophase state an urgent task. Para-substituted benzoic acids can form LCs through noncovalent dimerization into homodimers via hydrogen bonds, whose strength and, consequently, the temperature region of the mesophase state can be potentially altered by creating asymmetric heterodimers from different acids. This work investigates equimolar blends of p-n-alkylbenzoic (kBA, where k is the number of carbon atoms in the alkyl radical) and p-n-alkyloxybenzoic (kOBA) acids by calorimetry and viscometry to establish their phase transitions and regions of mesophase existence. Non-symmetric dimerization of acids leads to the extension of the nematic state region towards low temperatures and the appearance of new monotropic and enantiotropic phase transitions in several cases. Moreover, the crystal-nematic and nematic-isotropic phase changes have a two-step character for some acid blends, suggesting the formation of symmetric and asymmetric associates from heterodimers. The mixing of 6BA and 8OBA most strongly extends the region of the nematic state towards low temperatures (from 95-114 °C and 108-147 °C for initial homodimers, respectively, to 57-133 °C for the resulting heterodimer), whereas the combination of 4OBA and 5OBA gives the most extended high-temperature nematic phase (up to 156 °C) and that of 6BA and 9OBA (or 12OBA) provides the existence of a smectic phase at the lowest temperatures (down to 51 °C).

The Mechanisms of GPR55 Receptor Functional Selectivity during Apoptosis and Proliferation Regulation in Cancer Cells.

GPR55 is a non-canonical cannabinoid receptor, important for cancer proliferation. Depending on the ligand, it induces either cell proliferation or death. The objective of the study was to establish the mechanisms of this multidirectional signaling. Using the CRISPR-Cas9 system, the GPR55, CB1, CB2, and GPR18 receptor knockouts of the MDA-MB-231 line were obtained. After the CB2 receptor knockout, the pro-apoptotic activity of the pro-apoptotic ligand docosahexaenoyl dopamine (DHA-DA) slightly increased, while the pro-proliferative activity of the most active synthetic ligand of the GPR55 receptor (ML-184) completely disappeared. On the original cell line, the stimulatory effect of ML-184 was removed by the CB2 receptor blocker and by GPR55 receptor knockout. Thus, it can be confidently assumed that when proliferation is stimulated with the participation of the GPR55 receptor, a signal is transmitted from the CB2 receptor to the GPR55 receptor due to the formation of a heterodimer. GPR18 was additionally involved in the implementation of the pro-apoptotic effect of DHA-DA, while the CB1 receptor is not involved. In the implementation of the pro-apoptotic action of DHA-DA, the elimination of Gα13 led to a decrease in cytotoxicity. The obtained data provide novel details to the mechanism of the pro-proliferative action of GPR55.

The Phenomenon of Self-Induced Diastereomeric Anisochrony and Its Implications in NMR Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique largely applied in the analysis of discrimination processes involving enantiomeric substrates and chiral agents, which can interact with the analyte either via covalent bonding or via formation of diastereomeric solvates. However, enantiodiscrimination has been observed, in some cases, even in the absence of any additional chiral selector. The reasons behind this phenomenon must be found in the capability of some chiral substrates to interact with themselves by forming diastereomeric solvates in solution that can generate nonequivalences in the NMR spectra of enantiomerically enriched mixtures. As a result, differentiation of enantiomers is observed, thus allowing the quantification of the enantiomeric composition of the mixture under investigation. The tendency of certain substrates to self-aggregate and to generate diastereomeric adducts in solution can be defined as Self-Induced Diastereomeric Anisochrony (SIDA), but other acronyms have been used to refer to this phenomenon. In the present work, an overview of SIDA processes investigated via NMR spectroscopy will be provided, with a particular emphasis on the nature of the substrates involved, on the interaction mechanisms at the basis of the phenomenon, and on theoretical treatments proposed in the literature to explain them.

CRISPR System-Linked Self-Assembling Nanoplatforms for Inspection and Screening of Gastric Cancer Stem Cells.

Cancer stem cells (CSCs) possess a high degree of plasticity, constituting a formidable challenge to identify and screen CSCs in situ with outstanding specificity and sensitivity. To overcome this limitation, a self-assembled heterodimer consisting of clustered regularly interspaced short palindromic repeats/Cas12a (named A-CCA) linkage is designed for in situ identification and screening of gastric CSCs (GCSCs) from gastric cancer cells (GCCs). In this system, the editable character of crRNA performs recognition of dual-targets in GCSCs, effectively boosting the specificity of identification, while the enzymatic reaction of Cas12a contributes meaningfully to the sensitivity of sensing, enabling in situ examination and screening of GCSCs. Specifically, the A-CCA nanoplatforms hybridized with ABCG 2 and ABCB 1 overexpress in GCSCs, which can generate heterodimers and simultaneously restore the function of trans-cleavage. At this time, the asymmetry of the heterodimer causes a circular dichroism signal, which together with the recovered fluorescence signal form a dual-signals output system that can further ensure the precision of screening GCSC. Therefore, fluorescence-enhanced GCSCs can be sorted out from GCCs by flow cytometry. Furthermore, GCSCs screened by this assay possess extremely aggressive tumorigenic efficiency, providing a fundamental research object for further developing CSC targeted drugs in vivo.

Activation of TLR2 heterodimers-mediated NF-κB, MAPK, AKT signaling pathways is responsible for Vibrio alginolyticus triggered inflammatory response in vitro.

Vibrio alginolyticus is an important zoonotic marine pathogenic bacterium. Previous studies on the mechanism of innate immune against V. alginolyticus infection have been limited to aquatic animals, however, how V. alginolyticus activates mammalian immune cells has not been fully clarified. Here, ELISA combined RT-qPCR assays were used to detect the secretion and transcription level of pro-inflammatory cytokines and TLRs during V. alginolyticus infection of mice peritoneal macrophages (PMϕs). Western blotting was used to explore the phosphorylation levels of p38, JNK, ERK, AKT and NF-κB protein. Immunofluorescence assay was used to determine the location of NF-κB protein. Inhibition assay was used to study the role of up-regulated TLR in activated signaling pathways and the role of these pathways in the release of pro-inflammatory cytokines. Our data showed that V. alginolyticus can up-regulate the expression levels of IL-1ß, IL-6, IL-12 and TNF-α in PMϕs. In addition, V. alginolyticus stimulation activated the phosphorylation of p38, JNK and ERK were TLR2 heterodimers-dependent, whereas inhibitors of SB203580 (p38), SCH772984 (ERK) and SP600125 (JNK) significantly reduced IL-1ß, IL-6, IL-12 and TNF-α production. We further revealed that V. alginolyticus activated the signaling pathways of AKT via TLR2 heterodimers. The inhibitor of MK-2206 2HCl (AKT) negatively regulated the IL-1ß, IL-6 and TNF-α release levels. Moreover, V. alginolyticus infection of PMϕs resulted in TLR2 heterodimers-mediated activation of NF-κB and induced translocation of phosphorylated NF-κB protein from the cytoplasm into the nucleus via IκBα degradation. V. alginolyticus induced IL-1ß, IL-6, IL-12 and TNF-α release were blocked by the specific NF-κB inhibitor, BAY 11-7082. Taken together, our results suggested that activation of the TLR2 heterodimers-mediated downstream signaling pathways NF-κB, MAPK and AKT is responsible for inflammatory response during Vibrio alginolyticus infection in vitro.

7-Methylxanthine Influences the Behavior of ADORA2A-DRD2 Heterodimers in Human Retinal Pigment Epithelial Cells.

INTRODUCTION: The goal of this study was to investigate the presence of ADORA2A-DRD2 heterodimers in human retinal pigment epithelial (RPE) cells; determine if 7-methylxanthine (7-MX), a nonselective adenosine receptor antagonist which was used to control myopia progression, can influence the behavior of RPE cells through the ADORA2A-DRD2 receptor pathway; and assess the changes in the expression of signaling molecules during cellular signal transduction. METHODS: Human RPE cells were cultured in vitro in the presence or absence of 7-MX. Cell proliferation was evaluated with the CCK-8 assay. Apoptosis and necrosis rates were determined by annexin V-FITC/propidium iodide staining and flow cytometry. Immunofluorescence and coimmunoprecipitation were used to examine the protein expression and colocalization of ADORA2A and DRD2 in RPE cells. ADORA2A and DRD2 were knocked down with small interfering RNAs (siRNAs). Changes in the protein expression of ERK1/2 and phospho-ERK1/2 (pERK 1/2), which are signaling molecules downstream of dopamine receptors, were evaluated by Western blot analysis. RESULTS: Immunofluorescence and coimmunoprecipitation showed that ADORA2A and DRD2 were colocalized in RPE cells. The expression of ADORA2A in RPE cells was inhibited by treatment with 50 µmol/L 7-MX for 48 h, and the expression of DRD2, ERK1/2, and pERK1/2 was increased after treatment with 50 µmol/L 7-MX for 48 h. After siRNA-mediated knockdown of DRD2 in RPE cells and further treatment with 50 µmol/L 7-MX for 48 h, the expression of DRD2 was nearly restored to the level observed in the native control. At the experimental concentrations, 7-MX and siRNAs did not affect the proliferation or apoptosis of human RPE cells. CONCLUSIONS: ADORA2A and DRD2 heterodimers were present in RPE cells. 7-MX may affect the behaviors of RPE cells through the ADORA2A-DRD2 receptor pathway. 7-MX is an inhibitor of ADORA2A receptors that can prevent inhibition of the DRD2 receptor pathway and increase DRD2 receptor pathway activity. This phenomenon may explain the mechanism of action through which 7-MX can control myopia progression.

Characterization of the activity, aggregation, and toxicity of heterodimers of WT and ALS-associated mutant Sod1.

Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in sod1Δ yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies.