Enhancing therapeutic efficacy in luminal androgen receptor triple-negative breast cancer: exploring chidamide and enzalutamide as a promising combination strategy.

Extensive exploration of the molecular subtypes of triple-negative breast cancer (TNBC) is critical for advancing precision medicine. Notably, the luminal androgen receptor (LAR) subtype has attracted attention for targeted treatment combining androgen receptor antagonists and CDK4/6 inhibitors. Unfortunately, this strategy has proven to be of limited efficacy, highlighting the need for further optimization. Using our center's comprehensive multiomics dataset (n = 465), we identified novel therapeutic targets and evaluated their efficacy through multiple models, including in vitro LAR cell lines, in vivo cell-derived allograft models and ex vivo patient-derived organoids. Moreover, we conducted flow cytometry and RNA-seq analysis to unveil potential mechanisms underlying the regulation of tumor progression by these therapeutic strategies. LAR breast cancer cells exhibited sensitivity to chidamide and enzalutamide individually, with a drug combination assay revealing their synergistic effect. Crucially, this synergistic effect was verified through in vivo allograft models and patient-derived organoids. Furthermore, transcriptomic analysis demonstrated that the combination therapeutic strategy could inhibit tumor progression by regulating metabolism and autophagy. This study confirmed that the combination of histone deacetylase (HDAC) inhibitors and androgen receptor (AR) antagonists possessed greater therapeutic efficacy than monotherapy in LAR TNBC. This finding significantly bolsters the theoretical basis for the clinical translation of this combination therapy and provides an innovative strategy for the targeted treatment of LAR TNBC.

CCL19+ dendritic cells potentiate clinical benefit of anti-PD-(L)1 immunotherapy in triple-negative breast cancer.

BACKGROUND: The extensive involvement of dendritic cells (DCs) in immune contexture indicates their potent value in cancer immunotherapy. Understanding DC diversity in patient cohorts may strengthen the clinical benefit of immune checkpoint inhibitors (ICIs). METHODS: Single-cell profiling of breast tumors from two clinical trials was performed to investigate DC heterogeneity. Multiomics, tissue characterization, and pre-clinical experiments were used to evaluate the role of the identified DCs in the tumor microenvironment. Four independent clinical trials were leveraged to explore biomarkers to predict ICI and chemotherapy outcomes. FINDINGS: We identified a distinct CCL19-expressing functional state of DCs associated with favorable responses to anti-programmed death (ligand)-1 (PD-(L)1), which displayed migratory and immunomodulatory phenotypes. These cells were correlated with antitumor T cell immunity and the presence of tertiary lymphoid structures and lymphoid aggregates, defining immunogenic microenvironments in triple-negative breast cancer. In vivo, CCL19+ DC deletion by Ccl19 gene ablation dampened CCR7+CD8+ T cells and tumor elimination in response to anti-PD-1. Notably, high circulating and intratumoral CCL19 levels were associated with superior response and survival in patients receiving anti-PD-1 but not chemotherapy. CONCLUSIONS: We uncovered a critical role of DC subsets in immunotherapy, which has implications for designing novel therapies and patient stratification strategies. FUNDING: This study was funded by the National Key Research and Development Project of China, the National Natural Science Foundation of China, the Program of Shanghai Academic/Technology Research Leader, the Natural Science Foundation of Shanghai, the Shanghai Key Laboratory of Breast Cancer, the Shanghai Hospital Development Center (SHDC), and the Shanghai Health Commission.

Hippocampus RNA Sequencing of Pentylenetetrazole-Kindled Rats and Upon Treatment of Novel Chemical Q808.

The expression of genes altered in epilepsy remains incomplete, particularly in the hippocampus, which exhibits exquisite vulnerability to epilepsy. Q808 is an innovation chemical compound that has potent anti-convulsant effect. Exploring its mechanism can not only explore the pathogenesis of epilepsy but also provide a theoretical basis for its clinical application. The present study aimed to use RNA sequencing (RNA-seq) to reveal the gene transcriptomic profile of chronic pentylenetetrazole (PTZ)-kindled seizure rats and the difference of the PTZ model rat before and after treatment with Q808. Quantitative real-time PCR (qRT-PCR) was performed to validate the RNA-seq results. The protein level was estimated with Western blot. Hippocampal transcriptomic analysis showed that 289 differentially expressed genes (DEGs) were confirmed in the PTZ-kindled seizure group compared with the vehicle control. Gene cluster analysis identified most of the DEGs linked to neuronal apoptosis, neurogenesis, neuronal projections, and neurotransmitter regulation. After analysis across the three groups, 23 hub genes and 21 pathways were identified, and qRT-PCR analysis confirmed that most of the mRNA levels of hub genes were consistent with the RNA-seq results. Q808 treatment increased the level of ACE, a GABA-related protein. Our analysis showed the comprehensive compendium of genes and pathways differentially expressed for PTZ-kindled seizure rats and upon Q808 treatment in PTZ-kindled seizure, which may provide a theoretical basis to explore the mechanism and unique efficacy of Q808 and the pathophysiology of epilepsy in the future.

Double Cascading Charge Transfer at Integrated Perovskite/Organic Bulk Heterojunctions for Extended Near-Infrared Photoresponse and Enhanced Photocurrent.

Nowadays, nearly 48.7% near-infrared (NIR) irradiation (>800 nm) of the full solar spectrum has actually not been fully utilized since the state-of-the-art perovskite film usually can only absorb the most UV-vis sunlight radiation. Herein, high efficiency integrated Cs0.15 FA0.85 PbI3 perovskite/organic bulk (PC61 BM:D18:Y6) heterojunction solar cells with enhanced low energy photon harvest until 931 nm and a high maintained open circuit voltage of 1.04 V is successfully obtained. In particular, the favorable double cascading charge transfer paths pave an interesting possibility to spatially separate electrons upon visible light excitation and holes upon NIR photon absorption simultaneously at interfaces, significantly suppressing non-radiative bimolecular recombination and reaching the photocurrent density as high as 27.48 mA cm-2 and power conversion efficiency of 20.31%. Besides, the strong hydrophobicity of the ternary organic film has effectively prevented ambient humidity penetration and improves the stability of the perovskite in the continuous aging test (humidity > 60%) compared with the control device. This work has opened a significantly new window to improve the NIR light harvest for next generation highly efficient solar cells with full spectrum response.

The effect of a novel anticonvulsant chemical Q808 on gut microbiota and hippocampus neurotransmitters in pentylenetetrazole-induced seizures in rats.

BACKGROUND: The gut microbiota can modulate brain function and behavior and is increasingly recognized as an important factor in mediating the risk of epilepsy and the effects of seizure interventions. Drug therapy is one of the factors that influence the composition of the intestinal microbiota. Q808 is an innovative chemical with strong anticonvulsant activity and low neurotoxicity. However, studies evaluating the effect of Q808 on gut microbial communities are lacking. In this study, we aimed to evaluate the anticonvulsant activity of Q808 on a pentylenetetrazol (PTZ)-induced seizure model and analyze and compare the intestinal microbiota composition of non-PTZ vehicle control group, the PTZ-induced seizure model rats with and without Q808, through 16S rDNA sequencing. Neurotransmitter levels in the hippocampus were quantitatively estimated using HPLC-MS. RESULTS: The results suggest that Q808 effectively alleviates seizures in chronic PTZ-kindled model rats. Additionally, based on the analyzed abundance of the gut microbiota, dysbacteriosis of model rats was found to be corrected after Q808 treatment at the phylum level. The unique bacterial taxa (e.g., Lactobacillus) that are associated with acetylcholine production, were significantly increased. Several short-chain fatty acids (SCFAs)-producing bacteria, including Roseburia, Alloprevptella, Prevotellaceae_NK3B31_group, Prevotellaceae_UCG-001, and Prevotella_9, were enriched. In the hippocampus, the contents of acetylcholine increased, whereas the levels of 3-methoxytyramine, glutamine, and 5-hydroxyindole acetic acid (5-HIAA) decreased after Q808 treatment. CONCLUSIONS: This study demonstrates that Q808 can be used to remodel the dysbiosis of the gut microbiome and influence neurotransmitter levels in the hippocampus of PTZ-induced seizure model rats. We hope that these novel findings prompt further research on the interaction between gut microbiota and seizures and the mechanism of Q808.

Fine Emission Tuning from Near-Ultraviolet to Saturated Blue with Rationally Designed Carbene-Based [3 + 2 + 1] Iridium(III) Complexes.

We designed and synthesized a new class of six phosphorescent [3 + 2 + 1] iridium(III) complexes [(pbib)Ir(C^C)CN] bearing a tridentate 1,3-bis(1-butylimidazolin-2-ylidene) phenyl N-heterocyclic carbene (NHC)-based pincer ligand (pbib), bidentate imidazole-based NHC ligands (C^C), and a monodentate cyano group and investigated their photophysical, electrochemical, and thermal stabilities and electroluminescent properties. The extended π-conjugation of the imidazole-based C^C ligand is found to be the key to fine-tune the emission energies from ultraviolet blue (λ = 378 nm) to saturated blue (λ = 482 nm), as shown by electrochemical and photophysical studies, which is also revealed by the density functional theory (DFT) and time-dependent DFT calculations. Vacuum-deposited organic light-emitting diode devices have been fabricated with these newly synthesized emitters and exhibited the best external quantum efficiency of 6.4% and Commission International de L'Éclairage (CIE) coordinates of (0.163, 0.096), where the CIE y is very similar to the National Television System Committee standard blue CIE (x, y) coordinates of (0.149, 0.085). These results indicate that the novel [3 + 2 + 1] coordinated iridium(III) complexes [(pbib)Ir(C^C)CN], having a saturated blue emission, not only could alleviate the photodegradation of the emitters when compared to [(pbib)Ir(pmi)CN] but also provide new design strategies of saturated-blue-emitting iridium(III) complexes.

Highly Efficient Phosphorescent Blue-Emitting [3+2+1] Coordinated Iridium (III) Complex for OLED Application.

Cyclometalated iridium (III) complexes are indispensable in the field of phosphorescent organic light-emitting diodes (PhOLEDs), while the improvement of blue iridium (III) complexes is as yet limited and challenging. More diversified blue emitters are needed to break through the bottleneck of the industry. Hence, a novel [3+2+1] coordinated iridium (III) complex (noted as Ir-dfpMepy-CN) bearing tridentate bis-N-heterocyclic carbene (NHC) chelate (2,6-bisimidazolylidene benzene), bidentate chelates 2-(2,4-difluorophenyl)-4-methylpyridine (dfpMepy), and monodentate ligand (-CN) has been designed and synthesized. The tridentate bis-NHC ligand enhances molecular stability by forming strong bonds with the center iridium atom. The electron-withdrawing groups in the bidentate ligand (dfpMepy) and monodentate ligand (-CN) ameliorate the stability of the HOMO levels. Ir-dfpMepy-CN shows photoluminescence peaks of 440 and 466 nm with a high quantum efficiency of 84 ± 5%. Additionally, the HATCN (10 nm)/TAPC (40 nm)/TcTa (10 nm)/10 wt% Ir-dfpMepy-CN in DPEPO (10 nm)/TmPyPB (40 nm)/Liq (2.5 nm)/Al (100 nm) OLED device employing the complex shows a CIE coordinate of (0.16, 0.17), reaching a deeper blue emission. The high quantum efficiency is attributed to rapid singlet to triplet charge transfer transition of 0.9-1.2 ps. The successful synthesis of Ir-dfpMepy-CN has opened a new window to develop advanced blue emitters and dopant alternatives for future efficient blue PhOLEDs.

Effect and mechanism of berberine against polycystic ovary syndrome.

For women of reproductive age, polycystic ovary syndrome (PCOS) is not a rare heterogeneous endocrine disorder and metabolic dysfunction. Menstrual problems, hyperandrogenism, polycystic ovary (PCO) and infertility often affect these women, and they are also prone to metabolic syndrome (MS) and insulin resistance (IR). As an isoquinoline alkaloid, Berberine (BBR) is the main effective component of Coptis. BBR, as a multi-target, multi-path plant extract, can interfere with the development of PCOS and relate to pathological process from many aspects, with less adverse reactions. It is mentioned in this review that BBR can alleviate IR, reduce the level of serum androgen, regulate lipid metabolism and moderate chronic inflammation. BBR is often used in combination with metformin, compound cyproterone (CPA) and other drugs, in order to achieve better therapeutic effect on PCOS.

Air stable and highly efficient Bi3+-doped Cs2SnCl6 for blue light-emitting diodes.

Cs2SnCl6 perovskite has recently attracted attention as a promising optoelectronic material owing to its better stability and reduced toxicity than its lead counterparts. However, its luminescence performance hardly satisfies the requirements. Hence, a series of Bi3+-doped Cs2SnCl6 (Cs2SnCl6:Bi3+) with enhanced luminescence were synthesized by a solution-phase route. The results show that the initial concentration of Sn2+ can adjust the nucleation density and the quality of the crystal nucleus growth, which will affect the Bi3+ doping amount, crystal morphology, and photophysical properties of Cs2SnCl6:Bi3+. Cs2SnCl6:Bi3+ shows excellent stability in the atmosphere with a photoluminescence (PL) of around 456 nm and a photoluminescence quantum yield (PLQY) of 31%. The luminescence performance results from [BiSn4+ 3+ + VCl] defects caused by the Bi3+ doping. The blue LED based on the Cs2SnCl6:Bi3+ phosphor exhibits a long life of about 120 h and a Commission Internationale de L'Eclairage (CIE) color coordinates of (0.14, 0.11). This work demonstrates a strategy for Bi-doped perovskites with good stability. This investigation will facilitate the development of Cs2SnCl6:Bi3+ for blue LED applications.

Microscopic anatomy and ultrastructure of the resin ducts of Ferula ferulaeoides (Steud.) Korov. in Xinjiang.

Ferula ferulaeoides (Steud.) Korov. is a perennial herb that belongs to Umbelliferae (Apiaceae). Its resin and roots have extensive commercial and medicinal value in the Xinjiang region. However, the resin-secreting resin ducts (RDs) of F. ferulaeoides have not been studied in detail. This study used optical and transmission electron microscopy to explore the anatomical features, including the distribution, size, and structure, of the RDs among different organs of F. ferulaeoides. The microstructure data revealed that the RDs consisted of a round lumen, a layer of secretory cells, and multiple layers of sheath cells. Notably, the RDs in stem were arranged alternatively in a multilayered ring with vascular bundles of three distinct sizes. The ultrastructural analysis revealed that organelles in the secretory cells potentially play important roles in resin secretion. Those data may be of great significance to understanding the anatomy of the RDs in Ferula L. and Umbelliferae.

A Bilayer 2D-WS2/Organic-Based Heterojunction for High-Performance Photodetectors.

Two-dimensional (2D) tungsten disulfide (WS2) has inspired great efforts in optoelectronics, such as in solar cells, light-emitting diodes, and photodetectors. However, chemical vapor deposition (CVD) grown 2D WS2 domains with the coexistence of a discontinuous single layer and multilayers are still not suitable for the fabrication of photodetectors on a large scale. An emerging field in the integration of organic materials with 2D materials offers the advantages of molecular diversity and flexibility to provide an exciting aspect on high-performance device applications. Herein, we fabricated a photodetector based on a 2D-WS2/organic semiconductor materials (mixture of the (Poly-(N, N'-bis-4-butylphenyl-N, N'-bisphenyl) benzidine and Phenyl-C61-butyric acid methyl ester (Poly-TPD/PCBM)) heterojunction. The application of Poly-TPD/PCBM organic blend film enhanced light absorption, electrically connected the isolated WS2 domains, and promoted the separation of electron-hole pairs. The generated exciton could sufficiently diffuse to the interface of the WS2 and the organic blend layers for efficient charge separation, where Poly-TPD was favorable for hole carrier transport and PCBM for electron transport to their respective electrodes. We show that the photodetector exhibited high responsivity, detectivity, and an on/off ratio of 0.1 A/W, 1.1 × 1011 Jones, and 100, respectively. In addition, the photodetector showed a broad spectral response from 500 nm to 750 nm, with a peak external quantum efficiency (EQE) of 8%. Our work offers a facile solution-coating process combined with a CVD technique to prepare an inorganic/organic heterojunction photodetector with high performance on silicon substrate.

Iterative Non-Negative Matrix Factorization Filter for Blind Deconvolution in Photon/Ion Counting.

A digital filter based on non-negative matrix factorization (NMF) enables blind deconvolution of temporal information from large data sets, simultaneously recovering both photon arrival times and the instrument impulse response function (IRF). In general, the measured digital signals produced by modern analytical instrumentation are convolved by the corresponding IRFs, which can complicate quantitative analyses. Common examples include photon counting (PC), chromatography, super resolution imaging, fluorescence imaging, and mass spectrometry. Scintillation counting, in particular, provides a signal-to-noise advantage in measurements of low intensity signals, but has a limited dynamic range due to pulse overlap. This limitation can complicate the interpretation of data by masking temporal and amplitude information on the underlying detected signal. Typical methods for deconvolution of the photon events require advanced knowledge of the IRF, which is not generally trivial to obtain. In this work, a sliding window approach was developed to perform NMF one pixel at a time on short segments of large (e.g., 25 million point) data sets. Using random initial guesses for the IRF, the NMF filter simultaneously recovered both the deconvolved photon arrival times and the IRF. Applying the NMF filter to the analysis of triboluminescence (TL) data traces of active pharmaceutical ingredients enabled discrimination between different hypothesized physical origins of the signal.

Correction to: Incidence, mortality, and temporal patterns of oropharyngeal cancer in China: a population-based study.

In the original publication of this article [1], the number of death cases in 'Materials and methods' section is incorrect.

Incidence, mortality, and temporal patterns of oropharyngeal cancer in China: a population-based study.

BACKGROUND: Thus far, the incidence, mortality, and temporal trend data of oropharyngeal cancers (OPC) in China were few. We estimated the incidence, mortality, and temporal patterns of OPC in China during 2008-2012 according to the data from 135 population-based cancer registries to better understand the epidemiological pattern of OPC and to provide more precise information for OPC control in China. METHODS: According to the data of diagnosed OPC reported to 135 cancer registries during 2008-2012, we calculated age-standardized rate of incidence and mortality by 2000 Chinese standard population (ASRIC and ASRMC) and by 1985 Segi's world standard population (ASRIW and ASRMW) by age, sex, and geographic regions; annual percentage changes of OPC incidence and mortality were calculated using Joinpoint trend analysis. RESULTS: ASRIW and ASRMW were 2.22/100,000 person-years and 0.94/100,000 person-years, respectively. The incidence and mortality in urban areas were higher than those in rural areas. ASRIC and ASRIW of males were higher than those of females. The overall ASRIC of OPC was significantly increased by 6.2% annually between 2003 and 2006 (P = 0.038), but remained stable between 2007 and 2012 (P = 0.392). ASRIC and ASRMC of males and in rural areas were significantly increased in the last decade (P < 0.05), but the rates of females remained stable during the same period (P > 0.05). CONCLUSIONS: Across multiple cancer registries in China, there was an obvious increase in OPC in the recent decade, especially for incidence and mortality of males and in rural areas, whereas the rates of females remained stable. A healthy lifestyle should be advocated and early diagnosis and early treatment of OPC should be enhanced.

Nasopharyngeal carcinoma incidence and mortality in China, 2013.

BACKGROUND: We estimated the incidence and mortality of nasopharyngeal carcinoma (NPC) in China in 2010 according to the data of 145 domestic population-based cancer registries in 2014, and no such reports since then. Hence, to further and better understand its epidemiology in China and to provide more precise scientific information for its control and prevention in China, we analyzed the NPC incidence and mortality of 255 domestic population-based cancer registries, and estimated the national rates in 2013 again. METHODS: NPC incidence and mortality data of 255 domestic cancer registries in 2013, accepted by the 2016 National Cancer Registry Annual Report, were collected and collated, and the indices of NPC such as the numbers of new cases and deaths, crude rates, age-standardized rates, and truncated rates of incidence and mortality were calculated and analyzed. The incidence and mortality in China and its constituent areas were estimated according to the national population in 2013. RESULTS: An estimated 42,100 new cases and 21,320 deaths were attributed to NPC in China in 2013, accounting for 1.14% of all new cancer cases and 0.96% of all cancer-related deaths that year in China. Crude incidence and mortality of NPC were 3.09/100,000 and 1.57/100,000, respectively. World age-standardized incidence and mortality were 2.17/100,000 and 1.08/100,000, respectively. The incidence and mortality of males were obviously higher than those of females and slightly higher in urban areas than in rural areas. Among seven Chinese administrative regions, NPC incidence and mortality were obviously higher in South China than in other regions and lowest in North China. Top 3 incidence and mortality provinces and registering areas all located in South China. The age-specific incidence and mortality rose quickly from age 25-29 and 35 to 39 years, respectively, peaked at different ages and varied by location. CONCLUSIONS: These results demonstrated that NPC incidence and mortality in China in 2013 were also at high levels worldwide, which suggested that its control and prevention should be enhanced.

A Reduced Graphene Oxide/Disodium Terephthalate Hybrid as a High-Performance Anode for Sodium-Ion Batteries.

As a promising candidate for large-scale energy storage systems, sodium-ion batteries (SIBs) are experiencing a rapid development. Organic conjugated carboxylic acid anodes not only have tailorable electrochemical properties but also are easily accessible. However, the low stability and electrical conductivity hamper their practical applications. In this study, disodium terephthalate (Na2 TP), the most favorable organic conjugated carboxylic acid anode material for SIBs, was proposed to integrate with graphene oxide (GO) by an anti-solvent precipitation process, which ensures the uniform and tight coating of GO on the Na2 TP surface. GO is electrochemically reduced during the first several cycles of the electrochemical measurement, which buffers the volume change and improves the electrical conductivity of Na2 TP, resulting in a better cyclic and rate performance. The incorporation of only 5 wt % GO onto Na2 TP leads to a reversible capability of 235 mA h g-1 after 100 cycles at a current rate of 0.1 C, which is the best among the state of the art organic anodes for SIBs. The one-step synthesis together with the low costs of the raw materials show a promise for the scalable preparation of anode materials for practical SIBs.

Female breast cancer incidence and mortality in China, 2013.

BACKGROUND: Breast cancer is the most common cancer among women. Population-based cancer registration data from the National Central Cancer Registry were used to analyze and evaluate the incidence and mortality rates in China in 2013, providing scientific information for cancer prevention and control. METHODS: Pooled data were stratified by area (urban/rural), gender, and age group. National new cases and deaths were estimated using age-specific rates and the corresponding population in 2013. The Chinese population in 2000 and Segi's world population were used to calculate age-standardized rates. RESULTS: The estimated number of new breast cancer cases was about 278 800 in China in 2013. The crude incidence, age-standardized rate of incidence by Chinese standard population, and age-standardized rate of incidence by world standard population were 42.02/100 000, 30.41/100 000, and 28.42/100 000, respectively. The estimated number of breast cancer deaths was about 64 600 in China in 2013. The crude mortality, age-standardized rate of mortality by Chinese standard population, and age-standardized rate of mortality by world standard population were 9.74/100 000, 6.54/100 000, and 6.34/100 000, respectively. Both incidence and mortality were higher in urban than in rural areas. Age-specific breast cancer incidence significantly increased with age, particularly after age 20, and peaked at 50-55 years, while age-specific mortality increased rapidly after 25 years, peaking at 85+ years. CONCLUSIONS: Breast cancer is the most common cancer in Chinese women, especially women in urban areas. Comprehensive measures are needed to reduce the heavy burden of breast cancer.

Incidence and mortality of liver cancer in China in 2011.

BACKGROUND: Liver cancer is a common cancer with poor prognosis in China. In this study, the national population-based cancer registration data were used to evaluate and analyze liver cancer incidence and mortality in China in 2011 and provide a reference for liver cancer prevention and control. METHODS: We collected and evaluated the incidence and mortality data of liver cancer in 2011 from 177 cancer registries with qualified data. These data were used in the final analysis including calculating crude, standardized, and truncated incidences and mortalities, and estimated new liver cancer cases and deaths using age-specific rates and the corresponding populations. The national census in 2000 and Segi's population were used for age-standardized rates. RESULTS: The estimates of new liver cancer cases and deaths were 355,595 and 322,416, respectively, in China in 2011. The crude incidence, age-standardized rate of incidence by Chinese standard population (ASRIC), and age-standardized rate of incidence by world standard population (ASRIW) of liver cancer were 26.39/100,000, 19.48/100,000, and 19.10/100,000, respectively; the crude mortality, age-standardized rate of mortality by Chinese standard population (ASRMC), and age-standardized rate of mortality by world standard population (ASRMW) of liver cancer were 23.93/100,000,17.48/100,000, and 17.17/100,000, respectively. The incidence and mortality were higher in rural areas than in urban areas and higher in males than in females. The age-specific incidence and mortality of liver cancer increased greatly with age, particularly after 30 years and peaked at 80-84 or 85+ years. CONCLUSIONS: Liver cancer is a common cancer in China, particularly for males and residents in rural areas. Targeted prevention, early detection, and treatment programs should be carried out.

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

AIM: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. METHODS: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. RESULTS: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. CONCLUSION: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.