Membrane Receptors Research Articles

PKCα Activation via the Thyroid Hormone Membrane Receptor Is Key to Thyroid Cancer Growth

Thyroid carcinoma (TC) is the most common endocrine neoplasia, with its incidence increasing in the last 40 years worldwide. The determination of genetic and/or protein markers for thyroid carcinoma could increase diagnostic precision. Accumulated evidence shows that Protein kinase C alpha (PKCα) contributes to tumorigenesis and therapy resistance in cancer. However, the role of PKCα in TC remains poorly studied. Our group and others have demonstrated that PKCs can mediate the proliferative effects of thyroid hormones (THs) through their membrane receptor, the integrin αvβ3, in several cancer types. We found that PKCα is overexpressed in TC cell lines, and it also appeared as the predominant expressed isoform in public databases of TC patients. PKCα-depleted cells significantly reduced THs-induced proliferation, mediated by the integrin αvβ3 receptor, through AKT and Erk activation. In databases of TC patients, higher PKCα expression was associated with lower overall survival. Further analyses showed a positive correlation between PKCα and genes from the MAPK and PI3K-Akt pathways. Finally, immunohistochemical analysis showed abnormal upregulation of PKCα in human thyroid tumors. Our findings establish a potential role for PKCα in the control of hormone-induced proliferation that can be explored as a therapeutic and/or diagnostic target for TC.

Magnetically driven hydrogel microrobots for enhancing the therapeutic effect of anlotinib on osteosarcoma

IntroductionOsteosarcoma, characterized by high mortality and disability rates, poses a significant challenge due to its complex genetic background and the absence of specific membrane receptors, which hinder effective targeted therapy. Active targeting has emerged as a promising approach to address this issue.MethodsIn this study, magnetically driven hydrogel robots (MMHR) were utilized to load and deliver drugs precisely to target sites. The drugs included SCR1481B16, a specific MET inhibitor proven to inhibit MET-driven tumor growth, and Anlotinib. The microrobots were designed to navigate under magnetic guidance, enhancing drug efficacy while minimizing damage to normal tissues.ResultsThe study explored the potential of MMHR loaded with SCR1481B16 and Anlotinib in the treatment of Anlotinib-resistant osteosarcoma. The microrobots were successfully designed and produced, demonstrating the ability to deliver drugs precisely to tumor sites. Evaluation of the microrobots showed an enhanced sensitivity of tumors to Anlotinib, providing new insights into the treatment of drug-resistant osteosarcoma.DiscussionTumors overexpressing MET often develop resistance to VEGFR-targeted drugs. The use of SCR1481B16 as a MET inhibitor in combination with Anlotinib, delivered by magnetically driven hydrogel microrobots, offers a novel strategy to overcome this resistance. However, further in-depth research and validation are required before the clinical application of this method can be considered.ConclusionIn conclusion, magnetically driven hydrogel microrobots loaded with SCR1481B16 provide a promising new strategy for enhancing the sensitivity of Anlotinib-resistant osteosarcoma, bringing hope for future clinical applications in the treatment of this challenging disease.

Abstract 4140121: Lipoprotein(a) Local Sybthesis And Plasma Uptake Via MDFS5 in Human Calcified Valve

Background: High circulating levels of lipoprotein(a) [Lp(a)] are associated with increased risk of calcific aortic stenosis (CAS). Targeting Lp(a) metabolism holds promise for therapeutic interventions. However, the precise mechanisms by which Lp(a) accumulates on aortic valves still not fully understood. Hypothesis: Lp(a) could be synthesized in situ in human aortic valves under specific stimuli, such as calcification, in addition to being taken up from plasma via membrane receptors, including the newly identified receptor Major Facilitator Superfamily Domain Containing 5 (MFSD5). Methods: Immunohistochemistry was used to detect the Lp(a) and its receptor MFSD5 in human aortic valves from CAS patients or controls, which were non-CAS patients. Plasma Lp(a) levels were measured in individuals undergoing aortic valve resection. Human valvular interstitial cells (VICs) were cultured in procalcifying medium, with or without Lp(a) (10uM) for 14 and 21 days. Von-kossa or Alizarin Red staining were used to assess valve calcification. Expression of Lp(a) and MFSD5 was analyzed with quantitative RT-PCR and western blot. Results: Lp(a) was nearly absent in control valve site, whereas it was abundantly present in valve site from CAS patients (p＜0.05). The higher the level of Lp(a) detected, the greater the extent of calcification noted in valve site. The mRNA and protein levels of Lp(a) extracted from calcified human valves were significantly higher compared to controls. However, the concentration of plasma Lp(a) did not affect the expression of Lp(a) in valve site (p＞0.05). In vitro studies showed that Lp(a) mRNA was minimally detectable in normal VICs, but was significantly upregulated following exposure to procalcifying medium (p < 0.05). Lp(a) incubation led to severer calcification than that induced by procalcifying medium alone (p < 0.05). As MFSD5 was a newly identified receptor of Lp(a), we also test its expression. MDFS5 was increased in human calcified valve (p＜0.05) and calcified VICs (p＜0.05). These results showed that the Lp(a) in valve could be increased by either uptake from serum or by synthesized locally by proper stimuli. Conclusion: Our study has shown that the degree of aortic valve calcification is correlated with the level of Lp(a) in the valve site rather than in serum. We also verified MFSD5 was the receptor of Lp(a) on human valve. Targeting Lp(a) accumulation at the valve site may offer a more effective strategy for the prevention or treatment of CAS.

Expression analysis of CsNAC30 gene in cucumber under drought stress

This study performed a bioinformatics analysis of the CsNAC30 gene and examined its expression levels in cucumber leaves and roots under drought stress induced by PEG-6000. The bioinformatics analysis showed that the open reading frame (ORF) of CsNAC30 is 1254 bp, encoding a protein consisting of 417 amino acids. This protein is relatively unstable and hydrophilic, lacks transmembrane regions, and contains 49 phosphorylation sites. It also features a conserved NAM domain at the N-terminus. Homology and phylogenetic analyses indicate that CsNAC30 is a member of the NAC transcription factor family and is most closely related to the melon CMeNAC105 and the winter melon BhNAC96. Quantitative real-time PCR (qPCR) revealed that CsNAC30 expression is induced by drought stress, with relative levels increasing in both leaves and roots following PEG treatment. Expression peaked at 6 h in leaves and 9 h in roots before decreasing. These findings suggest that CsNAC30 plays a role in the cucumber’s response to drought stress.

Tumor Cell-Specific Signal Processing Platform Controlled by ATP for Non-invasive Modulation of Cellular Behavior.

Regulating the spatial distribution of membrane receptors can artificially reprogram cellular behaviors, which play a critical biological role in various physiological and pathological processes. Herein, we construct a tumor cell-specific signal processing platform (TCS-SPP) for controlled promotion/inhibition of cellular-mesenchymal epithelial transition factor (c-Met) receptor dimerization to noninvasively modulate cellular behaviors. Upon the dual-aptamer recognition in the upstream input signal circuit (UISC) to discriminate target cancer cells, the membrane-anchored DNA signal processor (DSP) is activated for signal amplification via rolling circle amplification (RCA) followed by the working of an ATP molecular switch for signal conversion, achieving receptor modulation in the downstream output signal circuit (DOSC). Benefiting from the rigid structure of DSP, the protective effect, and spatial confinement effect of RCA products, this TCS-SPP has demonstrated good performance in accurately modulating cellular behavior such as cell migration, invasion, and proliferation, showing great potential for targeted cancer therapy and biomedical engineering applications.

Regulated N-glycosylation controls chaperone function and receptor trafficking.

One-fifth of human proteins are N-glycosylated in the endoplasmic reticulum (ER) by two oligosaccharyltransferases, OST-A and OST-B. Contrary to the prevailing view of N-glycosylation as a housekeeping function, we identified an ER pathway that modulates the activity of OST-A. Genetic analyses linked OST-A to HSP90B1, an ER chaperone for membrane receptors, and CCDC134, an ER luminal protein. During its translocation into the ER, an N-terminal peptide in HSP90B1 templates the assembly of a translocon complex containing CCDC134 and OST-A that protects HSP90B1 during folding, preventing its hyperglycosylation and degradation. Disruption of this pathway impairs WNT and IGF1R signaling and causes the bone developmental disorder osteogenesis imperfecta. Thus, N-glycosylation can be regulated by specificity factors in the ER to control cell surface receptor signaling and tissue development.

SLC2A1 variants cause late-onset epilepsy and the genetic-dependent stage feature

BackgroundThe SLC2A1 gene plays a vital role in brain energy metabolism. SLC2A1 variants have been reported to be associated with early-onset refractory seizures. This study aims to explore the association between the SLC2A1 gene and late-onset epilepsy.MethodsTrios-based whole-exome sequencing was performed on patients with epilepsy without acquired etiologies. The pathogenicity of the variants was assessed according to the American College of Medical Genetics and Genomics (ACMG) guidelines.ResultsA total of 14 heterozygous SLC2A1 variants were identified in 16 unrelated families. The variants were evaluated as “pathogenic” or “likely pathogenic” according to the ACMG guidelines. Ten cases (62.5%) presented with infantile onset seizures and developmental delay/intellectual disability and were diagnosed with developmental and epileptic encephalopathy (DEE). The other six cases (37.5%) exhibited late-onset seizures and normal development. They were diagnosed with idiopathic partial epilepsy (n = 2) or idiopathic generalized epilepsy (n = 4). Further analysis showed that DEE-associated variants tended to cluster in the transmembrane region, whereas the mild epilepsy-associated variants tended to locate in regions outside the transmembrane region, suggesting a potential molecular sub-regional effect. A total of 15 cases had delayed diagnosis, with the longest delay being 22 years. The SLC2A1 expression stage, which is expressed at relatively high level throughout the whole life span, from the embryonic to adult stages with two peaks at approximately four and 14 years, is generally consistent with the seizure onset age. In addition, patients with early-onset age had variants that were potentially associated with severe damage, suggesting a potential correlation between the age of disease onset and the damaging effects of the variants.ConclusionsSLC2A1 variants are associated with late-onset epilepsy, which is consistent with the genetic-dependent stage feature of SLC2A1. Early genetic diagnosis is important for treatment of patients with SLC2A1 variants.

Serum integrin accumulation during asthma exacerbation: The role of matrix metalloproteinases.

The inflammation caused by asthma exacerbation can lead to permanent changes in the airways and loss of lung function. Integrins are membrane receptors that interact with components of the extracellular matrix and cell adhesion molecules. It is known that these receptors can be found in soluble form in some conditions such as asthma, but it is unknown if exacerbation during asthma leads to soluble integrins. Our results indicated that asthma patients showed higher levels of soluble α1, α2, and β2 integrin subunits in their serum compared to controls, as confirmed by both ELISA and western blot. During asthma exacerbation, the levels of α2 and β2 integrin subunits increased even more compared to non-exacerbation and controls, while the α1 integrin subunit decreased. Western blot analysis identified two β2 integrin subunits, one at 75 kDa and another at 120 kDa; the 120 kDa subunit increased during asthma exacerbation. The activity of matrix metalloproteinase 9 (MMP9) increased during exacerbation, while MMP2 remained unchanged. Lower forced expiratory volume in 1 second (FEV1) values were associated with higher expression levels of α2, β1, and β2 integrin subunits. Active and latent MMP9 were correlated with the levels of the β2 integrin subunit, which means that at low levels of active and latent MMP9, there are lower levels of β2 integrin subunit. In conclusion, asthma exacerbation leads to the presence of soluble integrins, particularly the β2 subunit, most likely due to MMP9-induced proteolytic cleavage.

In situ cell-surface conformation of the TCR-CD3 signaling complex.

The extracellular molecular organization of the individual CD3 subunits around the αβ T cell receptor (TCR) is critical for initiating T cell signaling. In this study, we incorporate photo-crosslinkers at specific sites within the TCRα, TCRβ, CD3δ, and CD3γ subunits. Through crosslinking and docking, we identify a CD3ε'-CD3γ-CD3ε-CD3δ arrangement situated around the αβTCR in situ within the cell surface environment. We demonstrate the importance of cholesterol in maintaining the stability of the complex and that the 'in situ' complex structure mirrors the structure from 'detergent-purified' complexes. In addition, mutations aimed at stabilizing extracellular TCR-CD3 interfaces lead to poor signaling, suggesting that subunit fluidity is indispensable for signaling. Finally, employing photo-crosslinking and CD3 tetramer assays, we show that the TCR-CD3 complex undergoes minimal subunit movements or reorientations upon interaction with activating antibodies and pMHC tetramers. This suggests an absence of 'inactive-active' conformational states in the TCR constant regions and the extracellular CD3 subunits, unlike the transmembrane regions of the complex. This study contributes a nuanced understanding of TCR signaling, which may inform the development of therapeutics for immune-related disorders.

Integrin α2 in the microenvironment and the tumor compartment of digestive (gastrointestinal) cancers: emerging regulators and therapeutic opportunities

Integrins are a family of cell surface membrane receptors and play a crucial role in facilitating bidirectional cell signaling. Integrin α2 (ITGA2) is expressed across a range of cell types, including epithelial cells, platelets, megakaryocytes, and fibroblasts, where it functions as a surface marker and it is implicated in the cell movements. The most recent findings have indicated that ITAG2 has the potential to function as a novel regulatory factor in cancer, responsible for driving tumorigenesis, inducing chemoresistance, regulating genomic instability and remodeling tumor microenvironment. Hence, we primarily focus on elucidating the biological function and mechanism of ITGA2 within the digestive tumor microenvironment, while highlighting its prospective utilization as a therapeutic target for cancer therapy.

Activation of a GPCR, ORL1 Receptor: A Novel Therapy to Prevent Heart Failure Progression

The number of ischemic heart failure (HF) patients is growing dramatically worldwide. However, there are at present no preventive treatments for HF. Our previous study showed that Gata4 overexpression improved cardiac function after myocardial infarction in rat hearts. We also found that Gata4 overexpression significantly increased the expression of a Pnoc gene, an endogenous ligand for the cell membrane receptor ORL1. We hypothesized that the activation of the ORL1 receptor would suppress HF in a rat ischemic heart model. Adult Sprague Dawley rats (8 weeks old, six males and six females) underwent left anterior descending coronary artery ligation. Three weeks later, normal saline or MCOPPB (ORL1 activator, 2.5 mg/kg/day) intraperitoneal injection was started, and continued 5 days a week for 3 months. Echocardiography was performed six times: pre-operative, 3 days after coronary artery ligation, pre-MCOPPB or saline injection, and 1, 2, and 3 months after saline or MCOPPB injection started. Animals were euthanized after 3 months’ follow-up and the hearts were harvested for histological analysis. The ORL1 activator, MCOPPB, significantly improved cardiac function after myocardial infarction in rats (ejection fraction, MCOPPB vs. saline at euthanasia, 67 ± 3% vs. 43 ± 2%, p < 0.001). MCOPPB also decreased fibrosis and induced angiogenesis. Thus, the ORL1 activator, MCOPPB, may be a novel treatment for preventing HF progression.

The Role of Cholesterol in M2 Clustering and Viral Budding Explained.

The influenza A M2 homotetrameric channel consists of four transmembrane (TM) and four amphipathic helices (AHs). This viral proton channel is suggested to form clusters in the catenoid budding neck areas in raft-like domains of the plasma membrane, resulting in cell membrane scission and viral release. The channel clustering environment is rich in cholesterol. Previous experiments have shown that cholesterol significantly contributes to lipid bilayer undulations in viral buds. However, a clear explanation of membrane curvature from the distribution of cholesterol around the M2TM-AH clusters is lacking. Using coarse-grained molecular dynamics simulations of M2TM-AH in bilayers, we observed that M2 channels form specific, C2-symmetric, clusters with conical shapes driven by the attraction of their AHs. We showed that cholesterol stabilized the formation of M2 channel clusters by filling and bridging the conical gap between M2 channels at specific sites in the N-termini of adjacent channels or via the C-terminal region of TM and AHs, with the latter sites displaying a longer interaction time and higher stability. The potential of mean force calculations showed that when cholesterols occupy the identified interfacial binding sites between two M2 channels, the dimer is stabilized by 11 kJ/mol. This translates to the cholesterol-bound dimer being populated by almost 2 orders of magnitude compared to a dimer lacking cholesterol. We demonstrated that the cholesterol-bridged M2 channels can exert a lateral force on the surrounding membrane to induce the necessary negative Gaussian curvature profile, which permits spontaneous scission of the catenoid membrane neck and leads to viral buds and scission.

Distribution of progesterone receptors and the membrane component of progesterone receptor in various organs and tissues of male and female rats

Progesterone regulates reproductive processes and affects many functions of various non-reproductive organs. Its effects in mammals and humans are mediated by nuclear (nPRs) and membrane progesterone receptors (mPRs). The action of progesterone through different types of receptors may differ significantly and has tissue specific features. The expression of known types and subtypes of progesterone receptors in the tissues of male and female rats has been studied fragmentarily. The purpose of our work was to study the expression of five mPRs genes, as well as the nPRs gene and the membrane component of the progesterone receptor PGRMC I in the reproductive organs and in 17 non-reproductive tissues of male and female rats using reverse transcription followed by real-time PCR. In this study, it was shown that a high level of nPRs gene expression in rats is found not only in reproductive organs of females (uterus, ovary, mammary glands), but also in seminal vesicles of males, in the brain and trachea of both sexes, in blood vessels, and in the pancreas of females. The highest level of expression of mPRs genes of all subtypes was found in the testes, while expression of the gene encoding nPRs was practically undetectable in them. Expression of genes encoding mPRs was also detected in the liver and spleen of male and female rats, while expression of the gene encoding nPRs was at background levels. Virtually no expression of nPRs, mPRs, and membrane component of progesterone receptor (PGRMC I) genes was detected in muscle, and its level was very low in the heart in animals of both sexes. We found sex-specific differentiation of nuclear and membrane receptor mRNA levels in rats in non-reproductive tissues, characterized by a predominance of nPRs transcripts and three subtypes of mPRs (α, β, δ) in females and two subtypes of mPRs (γ, ε) in males. Data on the presence of progesterone receptors in tissues not involved in reproduction confirm the effect of progesterone on these organs. High levels of mRNA for various progesterone receptors in the tissues of male rats, such as the pancreas, lungs, kidney, and trachea, indicate an important physiological role of progestins not only in females, but also in males, which is still poorly understood. The work also discusses the known functions of progesterone receptors in the tissues studied.

Single-molecule imaging and molecular dynamics simulations reveal early activation of the MET receptor in cells

Embedding of cell-surface receptors into a membrane defines their dynamics but also complicates experimental characterization of their signaling complexes. The hepatocyte growth factor receptor MET is a receptor tyrosine kinase involved in cellular processes such as proliferation, migration, and survival. It is also targeted by the pathogen Listeria monocytogenes, whose invasion protein, internalin B (InlB), binds to MET, forming a signaling dimer that triggers pathogen internalization. Here we use an integrative structural biology approach, combining molecular dynamics simulations and single-molecule Förster resonance energy transfer (smFRET) in cells, to investigate the early stages of MET activation. Our simulations show that InlB binding stabilizes MET in a conformation that promotes dimer formation. smFRET reveals that the in situ dimer structure closely resembles one of two previously published crystal structures, though with key differences. This study refines our understanding of MET activation and provides a methodological framework for studying other plasma membrane receptors.

Transcriptomic Profile of Tef (Eragrostis tef) in Response to Drought.

The threat to world food security posed by drought is ever increasing. Tef [Eragrostis tef (Zucc.) Trotter] is an allotetraploid cereal crop that is a staple food for a large population in the Horn of Africa. While the grain of tef provides quality food for humans, its straw is the most palatable and nutritious feed for livestock. In addition, the tef plant is resilient to several biotic and abiotic stresses, especially to drought, making it an ideal candidate to study the molecular mechanisms conferring these properties. The transcriptome expression of tef leaf collected from plants grown under drought conditions was profiled using RNA-Seq and key genes were verified using RT-qPCR. This study revealed that tef exhibits a complex molecular network involving membrane receptors and transcription factors that regulate drought responses. We identified target genes related to hormones like ABA, auxin, and brassinosteroids and genes involved in antioxidant activity. The findings were compared to physiological measurements such as changes in stomatal conductance and contents of proline, chlorophyll and carotenoid. The insights gained from this work could play vital role in enhancing drought tolerance in other economically important cereals such as maize and rice.

Mucus-Penetrable Biomimetic Nanoantibiotics for Pathogen-Induced Pneumonia Treatment.

Bacterial pneumonia has garnered significant attention in the realm of infectious diseases owing to a surge in the incidence of severe infections coupled with the growing scarcity of efficacious therapeutic modalities. Antibiotic treatment is still an irreplaceable method for bacterial pneumonia because of its strong bactericidal activity and good clinical efficacy. However, the mucus layer forming after a bacterial infection in the lungs has been considered as the "Achilles' heels" facing the clinical application of such treatment. Herein, traceable biomimetic nanoantibiotics (BioNanoCFPs) were developed by loading indacenodithieno[3,2-b]thiophene (ITIC) and cefoperazone (CFP) in nanoplatforms coated with natural killer (NK) cell membranes. The BioNanoCFP exhibited excellent demonstrated mucus-penetrating abilities, facilitating their arrival at the infection site. The presence of Toll-like receptors in the NK cell membrane rendered the BioNanoCFP with the capability to recognize pathogen-associated molecular patterns within bacteria, allowing precise targeting of bacterial colonization sites and achieving substantial therapeutic efficacy. Overall, our findings demonstrate the viability and desirability of using NK cell membrane-mediated drug delivery as a promising strategy for precision treatment.

Symposium Review: The Impact of Fatty Acids as Bioactive Nutrients on the Development of Offspring

Fatty acids (FA) are normally considered a source of energy; however, some FA are essential nutrients with different biological functions such as ligands to membrane and nuclear receptors, and upon binding they modify cell function and transcript expression. The bioactive effects of the FA depend on the FA type and family (i.e., n-6 vs n-3). The FA effects on developmental programming have been studied in cattle and sheep with some similarities in the outcomes between species. Feeding n-3 FA during late gestation improves offspring production performance (i.e., milk yield in dairy cows and growth in beef cattle and sheep) compared with the offspring of dams supplemented with mono- and unsaturated FA or with offspring of dams with no FA supplementation. Also, there is a sexual dimorphism in the outcomes of n-3 FA supplementation, where the increase in growth due to n-3 FA seems to be more evident in males; but it might decrease growth in females. There are multiple assumptions as to how this physiological process occurs. Based on published literature, the developmental effect does not appear to be due to changes in hypothalamic regulations of dry matter intake and energy expenditure or liver and adipose tissue functions. The changes in offspring growth can be attributed to changes in gastrointestinal tract physiology, changes in immune response, or both, probably due to epigenetic changes in those tissues. Feeding n-3 FA in late gestation to the pregnant dam increases expression of amino acid transporters (mRNA and protein) in the offspring's duodenum, associated with changes in DNA methylation. Regarding immune function, the increase in offspring performance has been associated with decreased haptoglobin after weaning in calves or increases in lipid mediators, such as resolvin-D1 at birth. Omega-3 supplementation during late gestation affects offspring growth; changes in the offspring's gut and immune system biology can explain the sexual dysmorphism observed in changed body weight; however, we are unaware which of these basic mechanisms is responsible for the observed changes in biology.

Structural plasticity of arrestin-G protein coupled receptor complexes as a molecular determinant of signaling

G protein coupled receptors (GPCRs) are critically regulated by arrestins. In this study, high-resolution data was combined with molecular dynamics simulations to infer the determinants of β-arrestin 1 (βarr1)-GPCR coupling, using the V2 vasopressin receptor (V2R) as a model system.The study highlighted the extremely high plasticity of βarr1-GPCR complexes, dependent on receptor type, state, and membrane environment. The multiple functions of receptor-bound βarr1 are likely determined by the interplay of intrinsic flexibility and collective motions both as a bi-domain protein and as a whole. The two major collective motions of the whole βarr1, consisting in rotation parallel to the membrane plane and inclination with respect to the receptor main axis, are distinctly linked to the two intermolecular interfaces involved in tail and core interactions.The intermolecular dynamic coupling between βarr1 and V2R depends on the allosteric effect of the agonist arginine-vasopressin (AVP). In the absence of AVP the dynamic coupling concerns only tail interactions, while in the presence of AVP it involves both tail and core interactions. This suggests that constitutive and agonist-induced arrestin-receptor dynamic coupling is linked to distinct arrestin functions.

Ras-related Protein in Brain 4A (Rab4A) is Downregulated by miR-496 to inhibit the Progression of Gastric Cancer.

Ras-related protein in brain 4A (Rab4A), as a member of the Rab family, is involved in the intracellular circulation of membrane receptors or endocytic substances and regulates the progression of multiple tumors. From our results, the knockdown of Rab4A inhibited the proliferation, migration and invasion in AGS cells. Importantly, the surface expression of epidermal growth factor receptor (EGFR) declined significantly in Rab4A knockdown cells. The downstream pathway of EGFR was also inhibited after the transfection of Rab4A-specific siRNA, including AKT and β-catenin pathways. In addition, miR-496 down-regulated the expression of Rab4A in AGS cells. The result of the luciferase reporter assay showed that miR-496 could bind to the 3'UTR of Rab4A. In conclusion, the expression of Rab4A is inhibited by miR-496, and the knockdown of Rab4A inhibits the proliferation, migration and invasion through down-regulating the surface expression of EGFR. Rab4A is a potential target in the treatment of gastric cancer.

Electron tomography visualization of HIV-1 virions trapped by fusion inhibitors to host cells in infected tissues.

HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by ET. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by pre-hairpin intermediates. ET of HIV-1 pseudovirions bound to CD4+/CCR5+ TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow/liver/thymus mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study.IMPORTANCETrapped and untrapped HIV-1 virions, both mature and immature, were distinguished by localizing spokes via 3D tomographic reconstructions of HIV-1 infected and fusion-inhibitor-treated tissues of humanized mice. The findings of trapped HIV-1 virions in all tissues examined demonstrate a wide distribution of the CPT31 inhibitor, a desirable property for a potential therapeutic. In addition, the presence of virions trapped by spokes, particularly in vascular endothelial cells, demonstrates that the fusion inhibitors can be used as markers for potential HIV-1-target cells within tissues, facilitating the mapping of HIV-1 target cells within the complex cellular milieu of infected tissues.