Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

Structural basis of peptidomimetic agonism revealed by small- molecule GLP-1R agonists Boc5 and WB4-24.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3, and 7, one arm of both compounds was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts, showing an interaction feature substantially similar to the previously known small-molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by nonpeptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine-tuning of pharmacological efficacy in the development of future small-molecule therapeutics targeting GLP-1R.

The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1.

Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-Gi complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of ß-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine-receptor complexes, providing new insights into the mode of chemokine recognition.

Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry coupled with three-step data post-processing techniques for comprehensive profiling of the multiple components in Fufang Xianzhuli Ye.

INTRODUCTION: Fufang Xianzhuli (FXZL) Ye, a classical formula of traditional Chinese medicine, is composed of Succus Bambusae, Houttuyniae herba, Pinelliae Rhizoma, Zingiberis Rhizoma Recens, Eriobotryae Folium, Platycodonis Radix, and peppermint oil. For many years, FXZL has been primarily utilised in China to treat cough and phlegm. The chemical composition of FXZL has not been reported, which seriously affects the safety of the clinical application. OBJECTIVE: To establish a systematic method for rapidly classifying and recognising the chemical constituents in the FXZL for the safety of the clinical application. METHODS: An ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry coupled with a three-step data post-processing strategy was developed to screen the chemical constituents of FXZL. RESULTS: In this experiment, the diagnostic ions in FXZL were classified into six main compounds. A total of 106 compounds were unambiguously identified in FXZL based on their retention times, accurate masses, and tandem mass spectrometry data. These include 11 chlorogenic acids, three flavonoids, eight sesquiterpenoids, six organic acids, 65 triterpenoid saponins, and 13 other compounds. CONCLUSION: The chemical composition of FXZL was identified and summarised, providing useful information for quality control and a basis for further exploration of its active ingredients in vivo.

Effect of Chestnut (Castanea Mollissima Blume) Bur Polyphenol Extract on Shigella dysenteriae: Antibacterial Activity and the Mechanism.

Shigella dysenteriae is a highly pathogenic microorganism that can cause human bacillary dysentery by contaminating food and drinking water. This study investigated the antibacterial activity of chestnut bur polyphenol extract (CBPE) on S. dysenteriae and the underlying mechanism. The results showed that the minimum inhibitory concentration (MIC) of CBPE for S. dysenteriae was 0.4 mg/mL, and the minimum bactericidal concentration (MBC) was 1.6 mg/mL. CBPE treatment irreversibly disrupted cell morphology, decreased cell activity, and increased cell membrane permeability, cell membrane depolarization, and cell content leakage of S. dysenteriae, indicating that CBPE has obvious destructive effects on the cell membrane and cell wall of S. dysenteriae. Combined transcriptomic and metabolomics analysis revealed that CBPE inhibits S. dysenteriae by interfering with ABC protein transport, sulfur metabolism, purine metabolism, amino acid metabolism, glycerophospholipid metabolism, and some other pathways. These findings provide a theoretical basis for the prevention and treatment of S. dysenteriae infection with extract from chestnut burs.

Critical issues concerning the assessment sets in agreement assessment.

Since Bland and Altman's pioneering work, the assessment set has been an important constituent of agreement assessment. This article explores critical issues concerning the assessment set. First, we point out the fundamental differences between assessment approaches based on point estimation and those based on hypothesis testing related to the assessment set, and further present how the related disciplines choose between the two. Second, we argue that an assessment set with minimum volume should be preferred, as in the comparison of confidence intervals. Finally, we discuss assessment sets for the assessment of multiple methods, and propose two methods for the construction of assessment sets. Numerical examples further establish the applicability of these approaches.

Parametric and nonparametric improvements in Bland and Altman's assessment of agreement method.

The Bland-Altman method, which assesses agreement via an assessment set constructed by the difference of the measurement variables, has received great attention. Other assessment approaches have been proposed following the same difference-based framework. However, the exact assessment set constructed by the difference is achievable only for measurements with certain joint distributions. To provide a more general assessment framework, we propose two approaches. First, when the measurement distribution is known, we propose a parametric approach that constructs the assessment set through a measure of closeness corresponding to the distribution. Second, when the measurement distribution is unknown, we propose a nonparametric approach that constructs the assessment set through quantile regression. Both approaches quantify the degree of agreement with the presence of both systematic and random measurement errors, and enable one to go beyond the difference-based approach. Results of simulation and data analyses are presented to compare the two approaches.

Halorubrum depositum sp. nov., a Novel Halophilic Archaeon Isolated from a Salt Deposit.

A non-motile, pleomorphic rod-shaped or oval, red-pigmented (nearly scarlet), extremely halophilic archaeon, strain Y78T, was isolated from a salt deposit of Yunnan salt mine, China. Analysis of the 16S rRNA gene sequence showed that it was phylogenetically related to species of the genus Halorubrum, with a close relationship to Halorubrum rutilum YJ-18-S1T (98.6%), Halorubrum yunnanense Q85T (98.3%), and Halorubrum lipolyticum 9-3T (98.1%). The temperature, NaCl, and pH ranges for growth were 25-50 °C, 12-30% (w/v), and 6.5-9.0, respectively. Mg2+ was required for growth. The polar lipids of strain Y78T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and a sulfated diglycosyl diether. The DNA G+C content was 66.6 mol%. DNA-DNA hybridization values between strain Y78T and two closely related species of the genus Halorubrum were far below 70%. Based on the data presented in this study, strain Y78T represents a novel species for which the name Halorubrum depositum sp. nov. is proposed; the type strain is Y78T (= CGMCC 1.15456T = JCM 31272T).

A53T human α-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration.

In vitro evidence suggests that the inefficient removal of damaged mitochondria by macroautophagy contributes to Parkinson's disease (PD). Using a tissue-specific gene amplification strategy, we generated a transgenic mouse line with human α-synuclein A53T overexpression specifically in dopamine (DA) neurons. Transgenic mice showed profound early-onset mitochondria abnormalities, characterized by macroautophagy marker-positive cytoplasmic inclusions containing mainly mitochondrial remnants, which preceded the degeneration of DA neurons. Genetic deletion of either parkin or PINK1 in these transgenic mice significantly worsened mitochondrial pathologies, including drastically enlarged inclusions and loss of total mitochondria contents. These data suggest that mitochondria are the main targets of α-synuclein and their defective autophagic clearance plays a significant role during pathogenesis. Moreover, endogenous PINK1 or parkin is indispensable for the proper autophagic removal of damaged mitochondria. Our data for the first time establish an essential link between mitochondria macroautophagy impairments and DA neuron degeneration in an in vivo model based on known PD genetics. The model, its well-defined pathologies, and the demonstration of a main pathogenesis pathway in the present study have set the stage and direction of emphasis for future studies.

Loss of PINK1 attenuates HIF-1α induction by preventing 4E-BP1-dependent switch in protein translation under hypoxia.

Parkinson's disease (PD) has multiple proposed etiologies with implication of abnormalities in cellular homeostasis ranging from proteostasis to mitochondrial dynamics to energy metabolism. PINK1 mutations are associated with familial PD and here we discover a novel PINK1 mechanism in cellular stress response. Using hypoxia as a physiological trigger of oxidative stress and disruption in energy metabolism, we demonstrate that PINK1(-/-) mouse cells exhibited significantly reduced induction of HIF-1α protein, HIF-1α transcriptional activity, and hypoxia-responsive gene upregulation. Loss of PINK1 impairs both hypoxia-induced 4E-BP1 dephosphorylation and increase in the ratio of internal ribosomal entry site (IRES)-dependent to cap-dependent translation. These data suggest that PINK1 mediates adaptive responses by activating IRES-dependent translation, and the impairments in translation and the HIF-1α pathway may contribute to PINK1-associated PD pathogenesis that manifests under cellular stress.

In situ synthesis of luminescent dsDNA-Cu NCs stained with a dsDNA-lighted fluorophore for rapid and stable detection of histamine in food.

Poisonous histamine is accumulated in stale meat and fermented foods. The rapid and stable detection of histamine is essential for food safety. Herein, a ratiometric fluorometric method for histamine detection was designed through in situ preparing double-stranded DNA­copper nanoclusters (dsDNA-Cu NCs) stained with 4',6-diamidino-2-phenylindole (DAPI). dsDNA-Cu NCs with red emission were rapidly synthesized via mixing Cu2+, ascorbate and dsDNA at room temperature for 5 min. When DAPI was added during preparation, DAPI coordinated with the Cu element accompanied by the quenched red emission of dsDNA-Cu NCs, and DAPI bound to dsDNA together with the enhanced blue emission of DAPI. Upon adding DAPI and histamine simultaneously, the coordination of histamine with the Cu element further decreased the red emission of dsDNA-Cu NCs, and drove the movement of DAPI from the Cu element to dsDNA along with the enhanced blue emission of DAPI. Significantly, ratiometric fluorescence was insensitive to variations in instrument and environment, causing stable measurement. Meanwhile, in situ synthesis integrated probe preparation with analyte detection, reducing time consumption. Additionally, this method quantified histamine in the concentration range of 7-50 µM with a detection limit of 3.6 µM. It was applied to determining histamine in food with satisfactory accuracy and precision.

Association Between Genetically Determined Serum Corin and the Risk of Stroke in Chinese Adults: A Mendelian Randomization Study.

BACKGROUND: Serum corin has been associated with stroke in observational studies, but the underlying causality is uncertain. This study examined the causal association between corin and stroke through Mendelian randomization study. METHODS AND RESULTS: In the Gusu cohort, serum corin was assayed at baseline, and stroke incidents were prospectively obtained during 10 years of follow-up. Single-nucleotide polymorphisms (SNPs) in CORIN were genotyped by MassArray for 2310 participants (mean age, 53 years; 39% men). Seventeen SNPs passed the Hardy-Weinberg test and were considered the potential instruments. Only 1 SNP (rs2271037) determined variability of serum corin was significantly associated with stroke even after adjusting for conventional risk factors (hazard ratio [HR], 1.36 [95% CI, 1.00-1.85]). The weighted genetic risk score generated from the SNP-corin associations was significantly associated with stroke (HR, 2.01 [95% CI, 1.15-3.51]). Using this genetic risk score as the instrument, 1-sample Mendelian randomization analysis found a significant HR of stroke per-SD higher log2-transformed corin (HR, 1.37 [95% CI, 1.07-1.76]). The inverse variance-weighted analysis based on the SNP-corin and SNP-stroke associations found that the HR of stroke pre-SD higher log2-transformed corin was 5.92 (95% CI, 2.23-15.72). The effect estimates stayed consistent regardless of an individual SNP being removed from the instruments. An almost identical effect estimate was also confirmed by multiple other 2-sample Mendelian randomization methods. CONCLUSIONS: Genetically determined variations of serum corin concentration were significantly associated with the risk of stroke in Chinese adults. Elevated serum corin may be a risk factor for stroke.

PINK1 deficiency attenuates astrocyte proliferation through mitochondrial dysfunction, reduced AKT and increased p38 MAPK activation, and downregulation of EGFR.

PINK1 (PTEN induced putative kinase 1), a familial Parkinson's disease (PD)-related gene, is expressed in astrocytes, but little is known about its role in this cell type. Here, we found that astrocytes cultured from PINK1-knockout (KO) mice exhibit defective proliferative responses to epidermal growth factor (EGF) and fetal bovine serum. In PINK1-KO astrocytes, basal and EGF-induced p38 activation (phosphorylation) were increased whereas EGF receptor (EGFR) expression and AKT activation were decreased. p38 inhibition (SB203580) or knockdown with small interfering RNA (siRNA) rescued EGFR expression and AKT activation in PINK1-KO astrocytes. Proliferation defects in PINK1-KO astrocytes appeared to be linked to mitochondrial defects, manifesting as decreased mitochondrial mass and membrane potential, increased intracellular reactive oxygen species level, decreased glucose-uptake capacity, and decreased ATP production. Mitochondrial toxin (oligomycin) and a glucose-uptake inhibitor (phloretin) mimicked the PINK1-deficiency phenotype, decreasing astrocyte proliferation, EGFR expression and AKT activation, and increasing p38 activation. In addition, the proliferation defect in PINK1-KO astrocytes resulted in a delay in the wound healing process. Taken together, these results suggest that PINK1 deficiency causes astrocytes dysfunction, which may contribute to the development of PD due to delayed astrocytes-mediated repair of microenvironment in the brain.

Association between CORIN promoter methylation and stroke: Results from two independent samples of Chinese adults.

Objective: As the physical activator of natriuretic peptides, corin has been associated with stroke, but the underlying mechanism is not very clear. Here, we examined whether the CORIN promoter's methylation, an epigenetic DNA modification, was associated with the risk of stroke in two independent samples. Methods: A total of 1771 participants including 853 stroke cases and 918 healthy controls were included as a discovery sample and 2,498 community members with 10 years of follow-up were included as a replication sample. DNA methylation of the CORIN promoter was quantified by target bisulfite sequencing in both samples. We first examined the single CpG association, followed by a gene-based analysis of the joint association between multiple CpG methylation and stroke, adjusting for conventional risk factors. Results: The single CpG association analysis found that hypermethylation at all of the 9 CpG sites assayed was significantly associated with lower odds of prevalent stroke in the discovery sample (all p < 0.05), and three of them located at Chr4:47840038 (HR = 0.74, p = 0.015), Chr4:47839941 (HR = 0.80, p = 0.047), and Chr4:47839933 (HR = 0.82, p = 0.050) were also significantly associated with incident stroke in the replication sample. The gene-based association analysis found that DNA methylation of the 9 CpG sites at the CORIN promoter was jointly associated with stroke in both samples (all p < 0.05). Conclusion: DNA methylation levels of the CORIN gene promoter were lower in stroke patients and predicted a higher risk of incident stroke in Chinese adults. The underlying causality warranted further investigation.

Association between soluble neprilysin and diabetes: Findings from a prospective longitudinal study.

Background: The potential role of neprilysin (NEP) in glucose metabolism has been found by basic studies but lacks population evidence. The objective of this study was to examine the association between serum NEP and diabetes in Chinese adults. Methods: In a prospective longitudinal cohort study - the Gusu cohort (n=2,286, mean age: 52 years, 61.5% females), the cross-sectional, longitudinal, and prospective associations between serum NEP and diabetes were systemically examined by logistic regression adjusting for conventional risk factors. Serum NEP was measured at baseline using commercial ELISA assays. Fasting glucose was repeatedly measured 4 years apart. Results: The cross-sectional analysis found a positive association between serum NEP and fasting glucose at baseline (ß=0.08, P=0.004 for log-transformed NEP). This association persisted after controlling for the dynamic risk profiles during follow-up (ß=0.10, P=0.023 for log-transformed NEP). The prospective analysis found that a higher level of serum NEP at baseline was associated with a higher risk of diabetes during follow-up (OR=1.79, P=0.039 for log-transformed NEP). Conclusions: Serum NEP was not only associated with prevalent diabetes but also predicted the future risk of diabetes development in Chinese adults, independent of many behavioral and metabolic factors. Serum NEP may be a predictor and even a new therapeutic target for diabetes. However, the casualty and mechanisms of NEP in the development of diabetes require further investigation.

Deconstructing the role of MALAT1 in MAPK-signaling in melanoma: insights from antisense oligonucleotide treatment.

The long non-coding RNA (lncRNA) MALAT1 is a regulator of oncogenesis and cancer progression. MAPK-pathway upregulation is the main event in the development and progression of human cancer, including melanoma and recent studies have shown that MALAT1 has a significant impact on the regulation of gene and protein expression in the MAPK pathway. However, the role of MALAT1 in regulation of gene and protein expression of the MAPK-pathway kinases RAS, RAF, MEK and ERK in melanoma is largely unknown. We demonstrate the impacts of antisense oligonucleotide (ASO)-based MALAT1-inhibition on MAPK-pathway gene regulation in melanoma. Our results showed that MALAT1-ASO treatment decreased BRAF RNA expression and protein levels, and MALAT1 had increased correlation with MAPK-pathway associated genes in melanoma patient samples compared to healthy skin. Additionally, drug-induced MAPK inhibition upregulated MALAT1-expression, a finding that resonates with a paradigm of MALAT1-expression presented in this work: MALAT1 is downregulated in melanoma and other cancer types in which MALAT1 seems to be associated with MAPK-signaling, while MALAT1-ASO treatment strongly reduced the growth of melanoma cell lines, even in cases of resistance to MEK inhibition. MALAT1-ASO treatment significantly inhibited colony formation in vitro and reduced tumor growth in an NRAS-mutant melanoma xenograft mouse model in vivo, while showing no aberrant toxic side effects. Our findings demonstrate new insights into MALAT1-mediated MAPK-pathway gene regulation and a paradigm of MALAT1 expression in MAPK-signaling-dependent cancer types. MALAT1 maintains essential oncogenic functions, despite being downregulated.

Orthosteric and allosteric modulation of human HCAR2 signaling complex.

Hydroxycarboxylic acids are crucial metabolic intermediates involved in various physiological and pathological processes, some of which are recognized by specific hydroxycarboxylic acid receptors (HCARs). HCAR2 is one such receptor, activated by endogenous ß-hydroxybutyrate (3-HB) and butyrate, and is the target for Niacin. Interest in HCAR2 has been driven by its potential as a therapeutic target in cardiovascular and neuroinflammatory diseases. However, the limited understanding of how ligands bind to this receptor has hindered the development of alternative drugs able to avoid the common flushing side-effects associated with Niacin therapy. Here, we present three high-resolution structures of HCAR2-Gi1 complexes bound to four different ligands, one potent synthetic agonist (MK-6892) bound alone, and the two structures bound to the allosteric agonist compound 9n in conjunction with either the endogenous ligand 3-HB or niacin. These structures coupled with our functional and computational analyses further our understanding of ligand recognition, allosteric modulation, and activation of HCAR2 and pave the way for the development of high-efficiency drugs with reduced side-effects.

The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impacton normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.