Endogenous cell membrane interactome mapping for the GLP-1 receptor in different cell types.

The GLP-1 receptor, one of the most successful drug targets for the treatment of type 2 diabetes and obesity, is known to engage multiple intracellular signaling proteins. However, it remains less explored how the receptor interacts with proteins on the cell membrane. Here, we present a ligand-based proximity labeling approach to interrogate the native cell membrane interactome for the GLP-1 receptor after agonist simulation. Our study identified several unreported putative cell membrane interactors for the endogenous receptor in either a pancreatic ß cell line or a neuronal cell line. We further uncovered new regulators of GLP-1 receptor-mediated signaling and insulinotropic responses in ß cells. Additionally, we obtained a time-resolved cell membrane interactome map for the receptor in ß cells. Therefore, our study provides a new approach that is generalizable to map endogenous cell membrane interactomes for G-protein-coupled receptors to decipher the molecular basis of their cell-type-specific functional regulation.

Protect the recurrent laryngeal nerves in US-guided microwave ablation of thyroid nodules at Zuckerkandl tubercle: a pilot study.

BACKGROUND: To evaluate the safety and efficacy of US-guided microwave ablation in patients with thyroid nodules at Zuckerkandl tubercle. METHODS: 103 consecutive patients with thyroid nodules at Zuckerkandl tubercle (ZTTN) were enrolled in this study from November 2017 to August 2021. Prior to the surgery or US-guided microwave ablation (MWA), preoperative ultrasound visualization of the recurrent laryngeal nerve (RLN) and ZTTN was performed, the size and the position relationship between them were observed. Patients were followed up at 1, 3, 6, and 12 months after MWA and the volume reduction rates (VRR) of the thyroid nodules were analyzed. RESULTS: All patients successfully had the RLN and ZTTN detected using ultrasound before surgery or ablation with a detection rate of 100%. For the 103 patients, the majority of ZTTN grades were categorized as grade 2, with the distance from the farthest outside of ZTTN to the outer edge of thyroid ranging between 6.0 and 10.0 mm. The position relationship between ZTTN and RLN was predominantly type A in 98 cases, with type D observed in 5 cases. After MWA, the median nodule volume had significantly decreased from 4.61 (2.34, 8.70) ml to 0.42 (0.15, 1.41) ml and the VRR achieved 84.36 ± 13.87% at 12 months. No nodules regrew throughout the 12-month follow-up period. Of the 11 patients experienced hoarseness due to RLN entrapment before ablation, 7 recovered immediately after separation of the RLN and ZTTN during MWA, 2 recovered after one week, and the other 2 recovered after two months. CONCLUSIONS: The RLN is closely related to ZTTN and mainly located at the back of ZTTN. The RLN can be separated from ZTTN by hydrodissection during MWA. US-guided MWA is a safe and effective treatment for ZTTN.

Ultrasound-guided percutaneous thermal ablation of parotid tumors: experience from two-centers.

Objective: To evaluate the efficacy and feasibility of ultrasound-guided percutaneous thermal ablation (TA) for treating benign parotid tumors.Methods: Patients with benign parotid tumors who underwent ultrasound-guided microwave ablation (MWA) or radiofrequency ablation (RFA) between January 2020 and March 2023 were included in this retrospective study. Change in tumor size (maximum diameter, tumor volume(V), volume reduction rate (VRR)) and cosmetic score (CS) were evaluated during a one-year follow-up period. We also recorded the incidence of any complications associated with TA.Results: A total of 23 patients (13 males and 10 females; median age 65 years, range 5-91 years) were included. The mean VRR at 1, 3, 6, and 12 months after TA was 37.03%±10.23%, 56.52%±8.76%, 82.28%±7.89%, and 89.39%±6.45%, respectively. Mean CS also changed from 3.39 ± 0.66 to 1.75 ± 0.93 (p < 0.001) by the end of follow-up time. Subgroup analysis showed that tumors with smaller initial maximum diameter had a faster CS reduction rate than those with larger initial diameter. The incidence of facial nerve dysfunction was 8.70%.Conclusion: Ultrasound-guided percutaneous TA is an effective and safe treatment option for patients with benign parotid tumors.

Prevalence of sarcopenic obesity: comparison of different diagnostic criteria and exploration of optimal screening methods.

BACKGROUND AND AIMS: Despite concerns on the adverse health outcomes of sarcopenic obesity (SO), exploration regarding the applicability of different diagnostic criteria and the optimal screening methods is still lacking. This study aims to compare the prevalence and diagnostic agreement of SO under four diagnostic criteria in Chongming, Shanghai, China and assess the diagnostic value of nine screening methods for SO. METHODS: The study population included older people aged ≥ 65 years. The Asian Working Group for Sarcopenia-2019 (AWGS-2019) was used to diagnose sarcopenia. Obesity was defined using percentage of body fat (PBF), percent of body fat exceeding the 60th percentile (60% PBF), body mass index (BMI) and waist circumference (WC). The four diagnostic criteria for SO were AWGS + PBF, AWGS + 60% PBF, AWGS + BMI and AWGS + WC. Nine screening methods were the sarcopenia questionnaire [the questionnaire with five items to screen for sarcopenia (SARC-F), the addition of calf circumference to the SARC-F (SARC-CalF), and the addition of elderly age and BMI to the SARC-F (SARC-F + EBM)] combined with commonly used obesity indicators. Cohen's kappa compared agreement between diagnostic criteria, whilst sensitivity, specificity, receiver operating characteristics (ROC) and area under the ROC curve (AUC) compared the diagnostic value of nine screening methods. RESULTS: A total of 1407 older people were enrolled. The prevalence of SO ranged from 0.3 to 9.9%. The highest agreement between AWGS + 60% PBF and AWGS + PBF. When the AWGS + PBF was used as the 'gold standard' (due to its high agreement and high prevalence), SARC-CalF + PBF had the highest AUC value, and SARC-F + BMI had the highest sensitivity. The recommended cut-off values for SARC-F + BMI are SARC-F ≥ 1 score and BMI ≥ 19.845 kg/m², and the recommended cut-off values for SARC-CalF + PBF are SARC-CalF ≥ 5 score and PBF ≥ 34.55%. CONCLUSION: The prevalence of SO varied greatly amongst the four diagnostic criteria. AWGS + PBF is recommended for diagnosing SO in older people. SARC-F + BMI and SARC-CalF + PBF can be used as screening methods for SO.

Ba3.5Cu7.55In1.15Se9: A Wide-Bandgap Copper Indium Selenide Reveals Strong Luminescence and Third-Harmonic Generation.

A new copper indium selenide, Ba3.5Cu7.55In1.15Se9, was synthesized by the KBr flux reaction at 800 °C. The compound crystallizes with orthorhombic Pnma, a = 46.1700(12) Å, b = 4.26710(10) Å, c = 19.8125(5) Å, and Z = 8. The structural framework mainly consists of four sites of cubane-type defective M4Se3 (M = Cu, Cu/In) units with disordered Cu+/In3+ ions present at the part corner of each unit. The single crystal emits intense photoluminescence at 657 nm with a relative quantum yield (RQY) 0.2 times that of rhodamine 6G powder. The compound belongs to a direct band gap at 1.91 eV, analyzed by Tauc's plot, and the energy is close to the PL position. The Hall effect measurement on a pressed pellet reveals an n-type conductivity with a carrier concentration of 3.358 × 1017 cm-3 and a mobility of 24.331 cm2 V-1 s-1. Furthermore, the compound produces a strong nonlinear third-harmonic generation (THG), with an χS(3) value of 1.3 × 105 pm2/V2 comparable to 1.6 × 105 pm2/V2 for AgGaSe2 measured at 800 nm.

Focused ultrasound-mediated blood-brain barrier opening combined with magnetic targeting cytomembrane based biomimetic microbubbles for glioblastoma therapy.

Glioblastoma is the most common type of brain tumor. Due to the presence of the blood-brain barrier, the effects of chemotherapy have been unsatisfactory. The combination of focused ultrasound and microbubbles to reversibly open the blood-brain barrier is now considered a key factor in improving treatment outcomes of glioblastoma. In this study, we developed bionic drug delivery microbubbles, which in combination with focused ultrasound had an obvious inhibitory effect on glioblastoma. We extracted the brain microvascular cell membranes, combined them with lipid components, and loaded them with superparamagnetic iron oxide and doxorubicin to prepare biomimetic drug delivery microbubbles (FeDOX@cellMBs). We demonstrated that FeDOX@cellMBs retained the intrinsic properties of loading, such as magnetic properties and drug toxicity, both in vitro and in vivo. FeDOX@cellMBs exhibited good tumor targeting and uptake under the combined action of magnetic and focused ultrasound. Importantly, the FeDOX@cellMBs demonstrated excellent internal stability and effectively inhibited tumor growth in orthotopic glioblastoma mice. Finally, organ H&E staining confirmed that FeDOX@cellMBs were safe for use. In conclusion, FeDOX@cellMBs successfully penetrated the blood-brain barrier and effectively inhibited glioblastoma growth under the combined effects of focused ultrasound and magnetic stimulation. These results provide a new approach for the treatment of glioblastoma, with implications for future clinical translation.

Comparing the efficacy of low-load resistance exercise combined with blood flow restriction versus conventional-load resistance exercise in Chinese community-dwelling older people with sarcopenic obesity: a study protocol for a randomised controlled trial.

INTRODUCTION: Sarcopenic obesity (SO) is characterised by decreased muscle mass, diminished muscle strength and/or reduced physical performance and a high percentage of body fat (PBF). Conventional-load resistance exercise (CRE) may be difficult for older people with SO owing to their declining physical functions. Low-load resistance exercise (LRE) combined with blood flow restriction (BFR; LRE-BFR) is a viable alternative to CRE for improving muscle mass and strength and potential exercise mode for managing SO. This study has two objectives: (1) to comprehensively evaluate the efficacy of CRE and LRE-BFR in improving body composition, muscle strength, physical performance, haematological parameters, cardiovascular disease (CVD) risk factors and quality of life and (2) to compare the efficacy of CRE and LRE-BFR and explore their potential mechanisms. METHODS AND ANALYSIS: This work is a 12-week assessor-blinded randomised clinical trial that will be conducted thrice a week. Sarcopenia will be defined using the Asian Working Group for Sarcopenia 2019, and obesity will be determined using the criteria developed by the World Health Organization. Community-dwelling older people aged ≥ 65 years will be screened as the participants using inclusion and exclusion criteria. A total of 33 participants will be randomised into a CRE group (n = 11), an LRE-BFR group (n = 11) and a control group that will be given only health education (n = 11). The primary outcomes will be knee extensor strength and PBF, and the secondary outcomes will be body composition, anthropometric measurements, muscle strength of upper limbs, physical performance, haematological parameters, CVD risk factors and quality of life. The outcomes will be measured at the baseline (week 0), end of the intervention (week 12) and follow up (week 24). All the collected data will be analysed following the intention-to-treat principle. ETHICS AND DISSEMINATION: The Ethics Research Committee has approved this study (approval No. CMEC-2022-KT-51). Changes or developments in this study will be reported at www.chictr.org.cn . TRIAL REGISTRATION: ChiCTR2300067296 (3 January 2023).

The role of parents' and friends' support in preventing suicide attempts among French sexual minority adolescents.

OBJECTIVES: French sexual minority adolescents are at higher risk for suicide attempts than their heterosexual peers. However, little is known about the role of parents' and friends' support among French lesbian, gay and bisexual (LGB) youth. This study aimed to research the role of their support in preventing suicide attempts among LGB adolescents in France. MATERIALS AND METHODS: Data were drawn from a French cross-sectional study entitled "Portraits d'adolescents". Parental support was defined by satisfactory relations between participants and their parents. Friends' support was defined by satisfactory relations between participants and their friends. Chi-square and multiple logistic regression analyses were used to estimate and identify associated factors of suicide attempts in LGB as opposed to heterosexual youth. RESULTS: Data from a sample of 14,265 French adolescents aged 13 to 20 were analyzed. Among them, 637 (4.47%) identified as LGB. Attempted suicide was independently associated with sexual orientation (30.7% vs 10.6%; OR = 2.59 [2.11-3.18]; p < 0.0001). Both parents' and friends' support appeared to be protective factors in suicide attempts among heterosexuals (adjusted ORs = 0.40 [0.35-0.46] and 0.61 [0.51-0.75], respectively), whereas in the LGB group, only parental support was significant (adjusted OR = 0.42 [0.27-0.65]), independently of other variables. DISCUSSION: Prevention efforts might be carried out by identifying within-group differences among French adolescents with different sexual orientations. The supportive role of family members should be strengthened. Positive resources and salutary support systems may effectively prevent suicide attempts. CONCLUSIONS: French LGB adolescents have a higher risk for suicide attempts than their heterosexual peers. Parental support was reconfirmed as a major protective factor against suicide attempts in sexual minority adolescents.

Impact of de-branched starch molecules and fatty acid complexes on the attenuation of ageing-induced cognitive impairment.

BACKGROUND: Ageing and associated cognitive impairments are becoming serious issues around the world. In this study, the physiological properties of three kinds of complexes of fatty acid (capric, stearic and oleic acid, respectively) and de-branched starch molecules were investigated via a d-galactose-induced ageing model. This study revealed differences in the regulation of cognitive impairment and brain damage following intervention of different complexes, which might highlight a potent approach for the prevention of this chronic disease. RESULTS: Data indicated that three complexes improved response time and cognitive function and the bio-parameter markers associated with oxidative stress in ageing rats. Among them, the complexes prepared from de-branched starch-oleic acid showed a greater improvement compared to others. In addition, de-branched starch-capric acid complex showed a higher improvement in the morphology of colon cells and hippocampal neuronal cells. The consumption of de-branched starch-capric acid and -oleic acid complexes generated more short-chain fatty acids in the gut. More importantly, the complexation of de-branched starch with either caprate or stearate enhanced gut Akkermansia. Therefore, it was proposed that the richness in Akkermansia and gut metabolites might be associated with reduced damage of the hippocampal neuronal cells induced by the ageing progress. Moreover, the AMPK (AMP-activated protein kinase) pathway was activated in liver in de-branched starch-capric acid complex diet. In summary, de-branched starch-capric acid complex exhibited a greater effect on the attenuation of ageing-induced cognitive impairment. CONCLUSION: This study might highlight a new approach for intervening in the cognitive impairment during the ageing progress via a food supply. © 2023 Society of Chemical Industry.

Saikosaponin A Inhibits Growth of Human Bladder Carcinoma T24 and 5637 Cells Both in Vitro and in Vivo.

Saikosaponin A (SSA)-a natural compound extracted from Radix bupleuri-possesses antitumor properties in several types of carcinomas. However, the role of SSA on bladder cancer and the mechanisms remain unclear. In this study, we have described the effect of SSA on human bladder cancer cell lines T24 and 5637 in the context of the regulation of mitochondrial pathways of apoptosis. In vitro, the Cell Counting Kit-8 (CCK-8) assay and cell wound healing assays were used to determine the proliferative effect of SSA treatment. Flow cytometry and Western blotting were performed to evaluate the apoptosis and related mechanisms. To further confirm that apoptosis is mediated through Caspase activation, Hoechst 33258 fluorescence staining assay was done after cells were treated with SSA and caspase inhibitor-Z-VAD-FMK. In vivo, an orthotopic xenograft mice model was adopted to evaluate the effect of SSA. The tumors were analyzed by hematoxylin-eosin (H&E) staining, immunohistochemical analysis, and Western blotting. In vitro, the results with CCK-8 assay showed obvious SSA-induced suppression in cell growth in a dose- and time-dependent manner. Flow cytometry analysis, Hoechst 33258 fluorescence staining assay and the assessment of the changes in the B-cell lymphoma 2 (Bcl-2) family protein expression level revealed that SSA could significantly induce cell apoptosis, which was associated with apoptosis via the mitochondrial pathways. In vivo, the results revealed a reduction in cell proliferation. In conclusion, our data suggest that SSA inhibits the growth of bladder cancer cells by activating the mitochondrial apoptosis pathway and inducing cell apoptosis.

Capturing RNA-protein interaction via CRUIS.

No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA-protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA-protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA-protein interactions in their natural environment, and provides new insights into RNA biology.

Effectiveness and safety of percutaneous kyphoplasty combined with zoledronic acid in treatment of osteoporotic vertebral compression fractures: a meta-analysis.

INTRODUCTION: To investigate the clinical effectiveness of combination treatment of percutaneous kyphoplasty (PKP) and zoledronic acid (ZOL) in the treatment of osteoporotic vertebral compression fracture (OVCF). MATERIALS AND METHODS: We searched studies investigating the PKP combined with ZOL in the treatment of OVCF. We used a fixed-effects or random-effects model to analyze the bone mineral density (BMD), visual analogue scale (VAS), Oswestry disability index (ODI), bone markers (N-MID, ß-CTX, and P1NP) and adverse events, expressed as weight mean difference (WMD) and risk ratio (RR) with 95% confidence interval (95% CI). RESULTS: We identified 5 cohort studies with a total of 440 patients. Compared with PKP alone, the combination treatment of PKP and ZOL significantly reduced the VAS score at 6 months (WMD = - 0.78, 95% CI - 1.42, - 0.14; P = 0.018), and 12 months (WMD = - 0.98, 95% CI - 1.46, - 0.51; P < 0.001). Moreover, the combination treatment also improved the BMD at 6 (WMD = 0.06, 95% CI 0.01, 0.11, P = 0.016) and 12 months (WMD = 0.20, 95% CI 0.03, 0.36, P = 0.018) after treatment. The ODI score in the combination group was significantly lower than in PKP group at 6, 12 and 24 months after treatment (at 6 months: WMD = - 9.25, 95% CI - 13.62, - 4.87 P < 0.001; at 12 months: WMD = - 9.21, 95% CI - 11.91, - 6.50, P < 0.001; at 24 months: WMD = - 7.26, 95% CI - 11.39, - 3.14, P = 0.001). The N-MID and P1NP values were found to be significantly lower in the combination group than the PKP group, but the ß-CTX value was similar between the two groups. There was no significant difference in incidence of adverse events between the two groups, but more adjacent vertebral fractures and bone cement leakage occurred in PKP alone group. CONCLUSION: In patients with OVCF, combination treatment of PKP and ZOL showed more effective than PKP alone in improving BMD and bone marker levels, relieving pain, as well as reducing the risk of new fractures. More large-scale RCTs are needed to verify our findings.

Understanding regional mobility resilience and its relationship with regional culture during the COVID-19 pandemic: A pathogen-stress theory perspective.

The global COVID-19 pandemic has severely impacted the passenger flow. Facing the same pandemic, various regions differ in the resilience of population mobility due to differences in the regional cultural. This study uses mobile big data to quantifies regional mobility resilience of 358 cities in China. Study results reveal the differences in regional mobility resilience of cities through spatial autocorrelation analysis, and verify the effects of regional culture on mobility resilience using a panel logit regression model based on pathogen-stress theory. Spatial heterogeneity and autocorrelation in the regional mobility resilience of Chinese cities are identified through spatial analysis, which are manifested by various hot spots over time. Moreover, the panel regression results indicate that the COVID-19 pandemic has a significant negative effect on regional mobility resilience; and that the negative effect of COVID-19 on regional mobility resilience is amplified in the cities with high degrees of dialect diversity, while it is weakened in the cities with high degrees of cultural tightness (which have strict norms and punishments for deviance). This study provides theoretical implications for mobility resilience in the context of COVID-19 and advances the pathogen-stress theory. Study findings also provide practical recommendations for regions to enhance regional mobility resilience under the challenges of future public health crisis events.

A proximity-tagging system to identify membrane protein-protein interactions.

The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein-protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.

Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.

Inhibitors of Apoptosis Protein (IAPs) are guardian ubiquitin ligases that keep classic proapoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5,000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2-conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates, allowing them to be efficiently purified for LC-MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes.

Protein tyrosine phosphatase receptor type R (PTPRR) antagonizes the Wnt signaling pathway in ovarian cancer by dephosphorylating and inactivating ß-catenin.

Despite a lack of mutations, accumulating evidence supports an important role for the Wnt/ß-catenin pathway in ovarian tumorigenesis. However, the molecular mechanism that contributes to the aberrant activation of the Wnt signaling cascade in ovarian cancer has not been fully elucidated. Here, we found that protein tyrosine phosphatase receptor type R (PTPRR) suppressed the activation of the Wnt/ß-catenin pathway in ovarian cancer. We performed an shRNA-based biochemical screen, which identified PTPRR as being responsible for tyrosine dephosphorylation of ß-catenin on Tyr-142, a key site controlling the transcriptional activity of ß-catenin. Of note, PTPRR was down-regulated in ovarian cancers, and ectopic PTPRR re-expression delayed ovarian cancer cell growth both in vitro and in vivo Using a proximity-based tagging system and RNA-Seq analysis, we identified a signaling nexus that includes PTPRR, α-catenin, ß-catenin, E-cadherin, and AT-rich interaction domain 3C (ARID3C) in ovarian cancer. Immunohistochemistry staining of human samples further suggested that PTPRR expression is inversely correlated with disease prognosis. Collectively, our findings indicate that PTPRR functions as a tumor suppressor in ovarian cancer by dephosphorylating and inactivating ß-catenin. These results suggest that PTPRR expression might have utility as a prognostic marker for predicting overall survival.

Spexin as an anxiety regulator in mouse hippocampus: Mechanisms for transcriptional regulation of spexin gene expression by corticotropin releasing factor.

Spexin (SPX) acts as a neuropeptide with pleiotropic functions that can participate in anxiety regulation. Corticotropin releasing factor (CRF) is widely expressed in brain tissues and associated with depression and anxiety and addiction. With the anxious mice under chronic unpredictable stress, we found SPX mRNA expression level in the hippocampus of the brain was significantly reduced, while local CRF mRNA expression level was increased. Furthermore, CRF injection in the hippocampus could also decrease SPX mRNA expression levels in hippocampus and other brain tissues, including pituitary and hypothalamus. With the primary mouse hippocampal cell model, CRF treatment could decrease SPX mRNA expression at hippocampal cell level and this inhibitory effect was mediated only by corticotropin releasing factor receptor 2 (CRFR2) but not corticotropin releasing factor receptor 1 (CRFR1). In HEK293 cells with CRFR2 over-expression, CRF could also inhibit SPX promoter activity coupling with AC/cAMP/PKA and MEK1/2/Erk1/2 cascades. In addition, Epac was also involved with the CRF-repressed SPX promoter activity and cross-talked with MEK1/2/Erk1/2 pathway. CRF could inhibit SPX gene expression in mouse hippocampus via transcriptional activation at the promoter level with coupling of AC/cAMP and MEK1/2/Erk1/2 signaling, which will be relevant to the anxiety response mediated by SPX in central nervous system.

Mutations That Increase the Stability of the Postfusion gp41 Conformation of the HIV-1 Envelope Glycoprotein Are Selected by both an X4 and R5 HIV-1 Virus To Escape Fusion Inhibitors Corresponding to Heptad Repeat 1 of gp41, but the gp120 Adaptive Mutations Differ between the Two Viruses.

Binding of the gp120 surface subunit of the envelope glycoprotein (Env) of HIV-1 to CD4 and chemokine receptors on target cells triggers refolding of the gp41 transmembrane subunit into a six-helix bundle (6HB) that promotes fusion between virus and host cell membranes. To elucidate details of Env entry and potential differences between viruses that use CXCR4 (X4) or CCR5 (R5) coreceptors, we generated viruses that are resistant to peptide fusion inhibitors corresponding to the first heptad repeat region (HR1) of gp41 that target fusion-intermediate conformations of Env. Previously we reported that an R5 virus selected two resistance pathways, each defined by an early gp41 resistance mutation in either HR1 or the second heptad repeat (HR2), to escape inhibition by an HR1 peptide, but preferentially selected the HR1 pathway to escape inhibition by a trimer-stabilized HR1 peptide. Here, we report that an X4 virus selected the same HR1 and HR2 resistance pathways as the R5 virus to escape inhibition by the HR1 peptide. However, in contrast to the R5 virus, the X4 virus selected a unique mutation in HR2 to escape inhibition by the trimer-stabilized peptide. Significantly, both of these X4 and R5 viruses acquired gp41 resistance mutations that improved the thermostability of the six-helix bundle, but they selected different gp120 adaptive mutations. These findings show that these X4 and R5 viruses use a similar resistance mechanism to escape from HR1 peptide inhibition but different gp120-gp41 interactions to regulate Env conformational changes.IMPORTANCE HIV-1 fuses with cells when the gp41 subunit of Env refolds into a 6HB after binding to cellular receptors. Peptides corresponding to HR1 or HR2 interrupt gp41 refolding and inhibit HIV infection. Previously, we found that a CCR5 coreceptor-tropic HIV-1 acquired a key HR1 or HR2 resistance mutation to escape HR1 peptide inhibitors but only the key HR1 mutation to escape a trimer-stabilized HR1 peptide inhibitor. Here, we report that a CXCR4 coreceptor-tropic HIV-1 selected the same key HR1 or HR2 mutations to escape inhibition by the HR1 peptide but different combinations of HR1 and HR2 mutations to escape the trimer-stabilized HR1 peptide. All gp41 mutations enhance 6HB stability to outcompete inhibitors, but gp120 adaptive mutations differed between these R5 and X4 viruses, providing new insights into gp120-gp41 functional interactions affecting Env refolding during HIV entry.

Mutations of Glu560 within HIV-1 Envelope Glycoprotein N-terminal heptad repeat region contribute to resistance to peptide inhibitors of virus entry.

BACKGROUND: Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of gp41 can inhibit HIV-1 infection in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that induces fusion between viral and host cell membranes. Previously, we found that HIV-1 acquired the mutations of Glu560 (E560) in HR1 of envelope (Env) to escape peptide inhibitors. The present study aimed to elucidate the critical role of position 560 in the virus entry and potential resistance mechanisms. RESULTS: The Glu560Lys/Asp/Gly (E560K/D/G) mutations in HR1 of gp41 that are selected under the pressure of N- and C-peptide inhibitors modified its molecular interactions with HR2 to change 6HB stability and peptide inhibitor binding. E560K mutation increased 6HB thermostability and resulted in resistance to N peptide inhibitors, but E560G or E560D as compensatory mutations destabilized the 6HB to reduce inhibitor binding and resulted in increased resistance to C peptide inhibitor, T20. Significantly, the neutralizing activities of all mutants to soluble CD4 and broadly neutralizing antibodies targeting membrane proximal external region, 2F5 and 4E10 were improved, indicating the mutations of E560 could regulate Env conformations through cross interactions with gp120 or gp41. The molecular modeling analysis of E560K/D/G mutants suggested that position 560 might interact with the residues within two potentially flexible topological layer 1 and layer 2 in the gp120 inner domain to apparently affect the CD4 utilization. The E560K/D/G mutations changed its interactions with Gln650 (Q650) in HR2 to contribute to the resistance of peptide inhibitors. CONCLUSIONS: These findings identify the contributions of mutations of E560K/D/G in the highly conserved gp41 and highlight Env's high degree of plasticity for virus entry and inhibitor design.

The structure-based traceless specific fluorescence labeling of the smoothened receptor.

The smoothened receptor (SMO) mediates the hedgehog (Hh) signaling pathway and plays a vital role in embryonic development and tumorigenesis. The visualization of SMO has the potential to provide new insights into its enigmatic mechanisms and associated disease pathogenesis. Based on recent progress in structural studies of SMO, we have designed and characterized a group of affinity probes to facilitate the turn-on fluorescence labeling of SMO at the ε-amine of K395. These chemical probes were derived from a potent SMO antagonist skeleton by the conjugation of a small non-fluorescent unit, O-nitrobenzoxadiazole (O-NBD). In this context, optimal probes were developed to be capable of efficiently and selectively lighting up SMO regardless of whether it is in micelles or in native membranes. More importantly, the resulting labeled SMO only bears a very small fluorophore and allows for the recovery of the unoccupied pocket by dissociation of the residual ligand module. These advantages should allow the probe to serve as a potential tool for monitoring SMO trafficking, understanding Hh activation mechanisms, and even the diagnosis of tumorigenesis in the future.