A capillary electrophoresis-based assay for carrier screening of the hotspot mutations in the CYP21A2 gene.

Molecular genetic analysis of the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene is challenging owing to the highly homologous with its pseudogene. A reliable approach for the large-scale population screening of CYP21A2 is required. This study aimed to establish and evaluate a capillary electrophoresis-based assay for hotspot mutation carrier screening of the CYP21A2 gene. A total of 22 different variants in the CYP21A2 gene were detected by a capillary electrophoresis-based assay consisting of single nucleotide primer extension (SNaPshot) and high-throughput ligation-dependent probe amplification (HLPA) in the Chinese population, and the results were validated by alternative methods. Among the 5376 subjects, 1.51 % (81/5376) individuals were identified as CYP21A2 pathogenic variant carriers, with a carrier rate of 1/66. A total of 11 different variants were identified, of which c.293-13A/C > G (33.33 %) was the most common variant, followed by c.844C > T (19.75 %), c.518T > A (19.75 %), and Del/Con (16.05 %). There was a 100 % concordance between capillary electrophoresis and alternative method results. Furthermore, a total of 63 individuals (1.17 %, 63/5376) carried the c.955C > T (p. Q319∗) variant, among which 61 (61/63, 96.83 %) had a duplicated CYP21A2 gene and are therefore not carriers of a CYP21A2 allele. In conclusion, the capillary electrophoresis-based assay is an accurate and effective approach for genotyping the CYP21A2 gene and has the potential for the large-scale population screening of CYP21A2.

Phenotypic, genetic, variation, and molecular function of CaMYB113 in pepper (Capsicum annuum L.).

Pepper (Capsicum annuum L.) is widely consumed vegetables worldwide, and F1 hybrids are highly sought after in the pepper seed industry. However, studies on gene mutations affecting the color of cotyledon are rare, and the same is true for peppers. In this study, a segregating population was developed by crossing the pepper accession 21C1344 with purple cotyledon and accession 21C912 with green cotyledon. Initially, a target genomic region was identified by screening polymorphic SSR markers distributed across 12 chromosomes. Subsequently, polymorphic markers were developed based on resequencing data from the two parental lines, and genetic linkage analysis was performed. This approach ultimately identified Capana10g001433 (CaMYB113) as the candidate gene responsible for the purple cotyledons. The gene mutation type in 21C912 represents a new mutation type distinct from the reported missense mutation types, and this mutation affects the biosynthesis of anthocyanins. Virus-induced gene silencing (VIGS) of CaMYB113 substantially decreased anthocyanin accumulation in the cotyledons. Subsequent overexpression of CaMYB113 resulted in purple callus and leaves of pepper, and changed the expression levels of downstream genes involved in anthocyanin synthesis. Yeast one-hybrid and dual-luciferase transient expression assays demonstrated the binding of CaMYB113 to anthocyanin biosynthesis-related genes, thereby regulating anthocyanin accumulation in pepper cotyledons.

Growing a single suspended perfect protein crystal in a fully noncontact manner.

Nucleation is a fundamental process that determines the structure, morphology, and properties of crystalline materials, and is difficult to control because it is unpredictable. Here, we demonstrate a new method to control the protein crystal nucleation using a magnetic force, where we manipulate the movement and coalescence of nucleation precursors by adding paramagnetic salt into the crystallization solution to constrain the number and position of nucleation. We found that protein nucleation could be significantly affected by the magnetic force that the gradient magnetic fields generate. When the magnetization force is sufficiently enough, nucleation can be confined to the crystallization solution with no interface contact; therefore, only one crystal nucleus appears, which results in noncontact suspension growth of a single crystal in the crystallization solution system. Under these situations, the nucleation rate significantly decreases due to the coalescence of the dense liquid phase, and the crystal growth rate also decreases due to the suppression of convection, which increases the crystal quality. Our findings provide a new method for the noncontact control of crystal nucleation and demonstrate that externally applied physical environments can be used to affect the liquid-liquid phase separation process.

Comparative single-cell multiome identifies evolutionary changes in neural progenitor cells during primate brain development.

Understanding the cellular and genetic mechanisms driving human-specific features of cortical development remains a challenge. We generated a cell-type resolved atlas of transcriptome and chromatin accessibility in the developing macaque and mouse prefrontal cortex (PFC). Comparing with published human data, our findings demonstrate that although the cortex cellular composition is overall conserved across species, progenitor cells show significant evolutionary divergence in cellular properties. Specifically, human neural progenitors exhibit extensive transcriptional rewiring in growth factor and extracellular matrix (ECM) pathways. Expression of the human-specific progenitor marker ITGA2 in the fetal mouse cortex increases the progenitor proliferation and the proportion of upper-layer neurons. These transcriptional divergences are primarily driven by altered activity in the distal regulatory elements. The chromatin regions with human-gained accessibility are enriched with human-specific sequence changes and polymorphisms linked to intelligence and neuropsychiatric disorders. Our results identify evolutionary changes in neural progenitors and putative gene regulatory mechanisms shaping primate brain evolution.

Bisphenol A Polyoxyethylene Ether Sodium Sulfate as a New Leveler for Electrodeposition of Copper.

The electrochemical behavior of three types of bisphenol A polyoxyethylene ether sodium sulfate (BPA) is investigated through cyclic voltammetry striping tests. The results indicate that BPA-25 exhibited the most significant inhibition of Cu deposition. The nanotwinned Cu (nt-Cu) films are prepared by direct current electrodeposition in an acidic Cu sulfate bath containing a combination of conventional bath additives. Transmission electron microscopy (TEM) and cross-sectional scanning electron microscopy (SEM) results revealed that nt-Cu consisted of microcolumnar grains with high-density nanoscale coherent twin boundaries oriented parallel to the growth surface. The interaction between BPA-25 and the poly2-sulfur-2-propane sodium sulfonate (SPS) was further analyzed through galvanostatic measurements (GMs) and electron backscatter diffraction (EBSD). The results demonstrate that BPA-25 and SPS underwent competitive adsorption on the cathode surface, leading to the formation of nt-Cu. This growth mechanism provides a new perspective for the preparation of nt-Cu by using direct current electrodeposition.

Single-cell landscape of long and short glandular trichomes in Nicotiana tabacum leaves.

Glandular trichomes (GTs) play a crucial role in plant defenses and the synthesis of secondary metabolites. Understanding the developmental trajectory of GTs is essential for unraveling their functional significance and potential applications. Here we established a comprehensive single-cell atlas of Nicotiana tabacum leaves, a model plant for GT studies. The atlas included a total of 40,433 cells and successfully captured both long GTs (LGTs) and short GTs (SGTs) from Nicotiana leaves. The developmental trajectories of these trichomes were delineated, revealing potential disparities in epidermal development. Comparative analysis of Arabidopsis and Nicotiana trichome development indicated limited similarity between Arabidopsis epidermal non-glandular trichomes and Nicotiana LGTs and SGTs, implying the essentiality of studying the genes directly involved in the development of Nicotiana GTs for a proper and comprehensive understanding of GT biology. Overall, our results provide profound insights into the developmental intricacies of the specialized GTs.

Delayed covering causes the accumulation of motile sperm, leading to overestimation of sperm concentration and motility with a Makler counting chamber.

According to the World Health Organization (WHO) manual, sperm concentration should be measured using an improved Neubauer hemocytometer, while sperm motility should be measured by manual assessment. However, in China, thousands of laboratories do not use the improved Neubauer hemocytometer or method; instead, the Makler counting chamber is one of the most widely used chambers. To study sources of error that could impact the measurement of the apparent concentration and motility of sperm using the Makler counting chamber and to verify its accuracy for clinical application, 67 semen samples from patients attending the Department of Andrology, West China Second University Hospital, Sichuan University (Chengdu, China) between 13 September 2023 and 27 September 2023, were included. Compared with applying the cover glass immediately, delaying the application of the cover glass for 5 s, 10 s, and 30 s resulted in average increases in the sperm concentration of 30.3%, 74.1%, and 107.5%, respectively (all P < 0.0001) and in the progressive motility (PR) of 17.7%, 30.8%, and 39.6%, respectively (all P < 0.0001). However, when the semen specimens were fixed with formaldehyde, a delay in the application of the cover glass for 5 s, 10 s, and 30 s resulted in an average increase in the sperm concentration of 6.7%, 10.8%, and 14.6%, respectively, compared with immediate application of the cover glass. The accumulation of motile sperm due to delays in the application of the cover glass is a significant source of error with the Makler counting chamber and should be avoided.

[Long-term efficacy of oblique lateral interbody fusion combined with vertebral screw fixation for the treatment of lumbar specific infection].

OBJECTIVE: To explore medium and long term efficacy of oblique lateral interbody fusion (OLIF) in treating lumbar specific infection. METHODS: From October 2017 to January 2021, 24 patients with lumbar specific infection were treated by OLIF combined with vertebral screw internal fixation, including 15 males and 9 females, aged from 27 to 61 years old with an average of (43.0±15.0) years old;the courses of disease ranged from 6 to 24 months with an average of (14.0±7.0) months;7 patients with L2-L3, 12 patients with L3-L4 and 5 patients with L4-L5;19 patients with tuberculosis infection and 5 patients with brucella infection. The amount of intraoperative blood loss, operative time and complications were recorded, and erythrocyte sedimentation rate(ESR), C-reactive protein (CRP), visual analogue scale (VAS), Japanese Orthopaedic Association(JOA) score and American Spinal Injury Association (ASIA) rating were compared before and one month after opertaion. RESULTS: All patients were followed up from 9 to 24 months with an average of (13.0±6.0) months. Operative time was (132.5±21.4) min, and intraoperative blood loss was (227.3±43.1) ml. ESR and CRP were decreased from (82.34±18.62) mmol·h-1 and (53.08±21.84) mg·L-1 before operation to (33.52±17.31) mmol·h-1 and (15.48±8.36) mg·L-1 at one month after operation, respectively (P<0.05). VAS was decreased from (7.52±1.36) before opertaion to (1.74±0.87) at one month after operation (P<0.05). JOA was increased from (17.86±3.95) before operation to (24.72±3.19) at one month after operation (P<0.05). Four patients had neurological symptoms before operation, and were classified to grade D according to ASIA classification, who were recovered to grade E at 1 month after operation. One patient was suffered from psoas major muscle injury after operation, and returned to normal at 3 weeks. One patient was suffered from abdominal distension and difficulty in defecation, and relieved after gastrointestinal decompression and enema. No complications such as abdominal organ injury and poor wound healing occurred in all patients. CONCLUSION: OLIF combined with vertebral screw internal fixation is a new minimally invasive surgical method for the treatment of lumbar specific infection, especially the lesion located on the middle lumbar vertebra. It has advantages of less trauma, short operation time, less blood loss, convenient operation, complete removal of the lesion, safety and effectiveness, and has good medium-and long-term efficacy for lumbar specific infection.

Single-Cell RNA-Seq Analysis of Hearts in Patients with Fetal Tetralogy of Fallot.

INTRODUCTION: To explore the cytological characteristics of tetralogy of Fallot (TOF), we collected samples and investigated the differences in the cytological classification between normal fetal hearts and fetal hearts with congenital defects. We then performed single-cell sequencing analysis to search for possible differential genes of disease markers. METHODS: Here, the right ventricles of a heart sample with TOF and a healthy human fetal heart sample were analyzed through single-cell sequencing. Data quality control filtering, comparison, quantification, and identification of recovered cells on the raw data were performed using Cell Ranger, thereby ultimately obtaining gene expression matrices for each cell. Subsequently, Seurat was used for cell filtration, standardization, cell subgroup classification, differential expression gene analysis of each subgroup, and marker gene screening. RESULTS: Bioinformatic analysis identified 9,979 and 15,224 cells from the healthy and diseased samples, respectively, with an average read depth of 25,000/cell. The cardiomyocyte cell populations, derived from the abnormal samples identified through the first-level graph-based analysis, were separated into six distinct cell clusters. CONCLUSION: Our study provides some information on TOF in a fetus, which can offer a new reference for the early detection and treatment of TOF by comparing defective heart cells with normal heart cells.

Sterile Diet Causes Gut Microbiome Collapse of Cancer Patients Post Hematopoietic Cell Transplantation, But Normal Diet Recovers Them.

Though sterile diet, post-transplantation surgery is a clinical strategy for patient care to prevent the infiltration of gut pathogens, less is known about its effects on the gut microbiome. Here, the gut microbiome dynamics of leukemia patients following a 120-day "sterile-normal" diet strategy posthematopoietic cell transplantation are examined. In contrast to the traditional idea, a sterile diet leads to the lowest gut microbiota diversity (p < 0.05) and short-chain fatty acids, promoted the proliferation of potential pathogens such as Streptococcus (up by 16.93%) and Lactobacillus (up by 40.30%), and 43.32% reduction in nodes and an 85.33% reduction in edges within the microbial interaction's network. Interestingly, a normal diet allows the gut microbiome recovery and significantly promotes the abundance of beneficial bacteria. These results indicate that a sterile diet leads to a collapse of the patient's gut microbiome and promoted the proliferation of potential pathogens. This assay is a starting point for a more sophisticated assessment of the effects of a sterile diet. The work also suggests a basic principle for the re-establishment of microbial equilibrium that supplementation of microbial taxa may be the key to the restoration of the degraded ecosystem.

Effectiveness and pregnancy outcomes of ultrasound-indicated and physical examination-indicated cervical cerclage: a retrospective study from a single centre.

OBJECTIVE: Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality worldwide, and cervical incompetence (CIC) is a significant contribution. Cervical cerclage (CC) is an effective obstetric intervention. However, many clinical factors affect the success rate of surgery. The objective was to investigate and compare the pregnancy and neonatal outcomes of patients who underwent ultrasound- and physical examination-indicated cervical cerclage and to explore the influencing factors of preterm delivery before 34 weeks. METHODS: The sociodemographic characteristics and clinical data of patients with a diagnosis of cervical incompetence who underwent ultrasound- and physical examination-indicated transvaginal cervical cerclage at Nanjing Maternal and Child Health Hospital from January 2020 to December 2022 were retrospectively analyzed. The pregnancy and neonatal outcomes of the patients were evaluated. Continuous variables were compared using Student's t test (for normally distributed data) or the Mann-Whitney U test (for nonnormally distributed data). Categorical variables were analysed using the chi-square test or Fisher's exact test. Additionally, logistic regression analyses and receiver operating characteristic curves were used to evaluate the associations of inflammatory markers with maternal and neonatal outcomes. RESULTS: This study included 141 participants who underwent cervical cerclage, including 71 with ultrasound-indicated cerclage and 70 with physical examination-indicated cerclage. Compared to those in the ultrasound-indicated cerclage group, the duration from cerclage to delivery, birth weight, and APGAR score in the physical examination-indicated cerclage group were significantly lower, and the rates of delivery at < 28 weeks, < 32 weeks, < 34 weeks, and < 37 weeks of gestation and neonatal mortality were significantly higher (all P < 0.05). Compared to those in the physical ultrasound-indicated cerclage group, in the physical examination-indicated cerclage group, maternal blood inflammatory markers, such as C-reactive protein (CRP), the systemic immune-inflammation index (SII) and the systemic inflammation response index (SIRI) were significantly higher (P < 0.05). Additionally, maternal blood inflammatory markers, such as the CRP, white blood cell count, platelet to lymphocyte ratio (PLR), SII, and SIRI were significantly higher in the group with delivery before 34 weeks of gestation. Furthermore, the results demonstrated that twin pregnancy had the highest OR for preterm delivery before 34 weeks of gestation (OR = 3.829; 95% CI 1.413-10.373; P = 0.008), as well as the following: the SII level (OR = 1.001; 95% CI 1.000-1.002; P = 0.003) and CRP level (OR = 1.083; 95% CI 1.038-1.131; P = 0.022). The risk factors for preterm delivery before 34 weeks of gestation were twin gestation, an increased SII level and an increased CRP level, which had good combined predictive value. CONCLUSION: In patients with cervical insufficiency, ultrasound-indicated cervical cerclage appears to lead to better pregnancy outcomes than physical examination-indicated cerclage. Twin pregnancy and maternal blood inflammatory markers, such as the CRP level and the SII, are associated with preterm delivery before 34 weeks of gestation.

Development of a 45K pepper GBTS liquid-phase gene chip and its application in genome-wide association studies.

Introduction: Pepper (Capsicum spp.) is a vegetable that is cultivated globally and has undergone extensive domestication, leading to a significant diversification in its agronomic traits. With the advancement of genomics in pepper and the reduction in sequencing costs, the high-throughput detection of single nucleotide polymorphisms (SNPs) and small insertions-deletions (indels) has become increasingly critical for analyzing pepper germplasms and improving breeding programs. As a result, there is a pressing need for a cost-effective, high-throughput, and versatile technique suitable for both foreground and background selection in pepper breeding. Methods: In the present study, Python-based web scraping scripts were utilized to systematically extract data from published literatures and relevant sequence databases focusing on pepper genomes. Subsequent to data extraction, SNPs and indels were meticulously identified and filtered. This process culminated in the delineation of core polymorphic sites, which were instrumental in the development of specific probes. Following this, comprehensive phenotypic and genotypic analyses were conducted on a diverse collection of 420 pepper germplasms. Concurrently, a genome-wide association study (GWAS) was conducted to elucidate the genetic determinants of helical fruit shape in peppers. Results: In this study, a 45K pepper Genotyping-By-Target-Sequencing (GBTS) liquid-phase gene chip was developed on the GenoBaits platform. This chip is composed of 45,389 probes, of which 42,535 are derived from core polymorphic sites (CPS) in the background genetic landscape, while 2,854 are associated with foreground agronomic traits, spanning across 43 traits. The CPS probes are spaced at an average interval of 68 Kb. We have assessed the performance of this chip on 420 pepper germplasms, with successful capture of target DNA fragments by 45,387 probes. Furthermore, the probe capture ratio surpassed 70% in 410 of the 420 germplasms tested. Using this chip, we have efficiently genotyped 273 germplasms for spiciness levels and elucidated the genetic relationships among 410 pepper germplasms. Our results allowed for precise clustering of sister lines and C. chinense germplasms. In addition, through a GWAS for helical fruit shape, we identified three quantitative trait loci (QTLs): heli2.1, heli11.1, and heli11.2. Within the heli11.1 QTL, a gene encoding the tubulin alpha chain was identified, suggesting its potential role in the helical growth pattern of pepper fruits. Discussion: In summary, the 45K pepper GBTS liquid-phase gene chip offers robust detection of polymorphic sites and is a promising tool for advancing research into pepper germplasm and the breeding of new pepper varieties.

MIB2 Functions in Oocyte Meiosis by Modulating Chromatin Configuration.

Chromatin configuration serves as a principal indicator of GV (germinal vesicle)-stage oocyte quality. However, the underlying mechanisms governing the chromatin configuration transition from NSN (non-surrounded nucleolus) to SN (surrounded nucleolus) remain unclear. In this study, by conducting a quantitative proteomic analysis, we identified an increased expression of the MIB2 (MIB E3 ubiquitin protein ligase 2) protein in SN oocytes. Specific depletion of MIB2 in SN oocytes not only leads to severe disruption of the meiotic apparatus and a higher incidence of aneuploidy but also adversely affects meiotic maturation and early embryo development. Notably, overexpression of MIB2 in NSN oocytes facilitates the chromatin configuration transition. Meantime, we observed that forced expression of MIB2 in NSN oocytes significantly mitigates spindle/chromosome disorganization and aneuploidy. In summary, our results suggest that chromatin configuration transition regulated by MIB2 is crucial for oocytes to acquire developmental competence.

CaAOS as a hub gene based on physiological and transcriptomic analyses of cold-resistant and cold-sensitive pepper cultivars.

The yield and quality of pepper are considerably influenced by the cold conditions. Herein, we performed morphological, physiological and transcriptomic analyses by using two pepper seedlings, '2379' (cold-resistant) and '2380' (cold-sensitive). Briefly, 60 samples from each cultivar were analyzed at four distinct time points (0, 6, 24 and 48 h) at 5 °C in darkness. The physiological indices and activities of enzymes exhibited marked differences between the two cultivars. Transcriptomic analysis indicated that, compared to the control group, 11,415 DEGs were identified in '2379' and '2380' at 24 h. In the early stage, the number of DEGs in '2379' was 5.68 times higher than that in '2380', potentially explaining the observed differences in tolerance to colds. Processes such as protein targeting to membranes, jasmonic acid (JA)-mediated signalling, cold response and abscisic acid-activated signalling were involved. Subsequently, we identified a hub gene, CaAOS, that is involved in JA biosynthesis, positively influences cold tolerance and is a target of CaMYC2. Variations in the GC-motif of the CaAOS's promoter may influence the expression levels of CaAOS under cold treatment. The result of this study may lead to the development of more effective strategies for enhancing cold tolerance, potentially benefitting pepper breeding in cold regions.

Spatiotemporal transcriptomic landscape of rice embryonic cells during seed germination.

Characterizing cellular features during seed germination is crucial for understanding the complex biological functions of different embryonic cells in regulating seed vigor and seedling establishment. We performed spatially enhanced resolution omics sequencing (Stereo-seq) and single-cell RNA sequencing (scRNA-seq) to capture spatially resolved single-cell transcriptomes of germinating rice embryos. An automated cell-segmentation model, employing deep learning, was developed to accommodate the analysis requirements. The spatial transcriptomes of 6, 24, 36, and 48 h after imbibition unveiled both known and previously unreported embryo cell types, including two unreported scutellum cell types, corroborated by in situ hybridization and functional exploration of marker genes. Temporal transcriptomic profiling delineated gene expression dynamics in distinct embryonic cell types during seed germination, highlighting key genes involved in nutrient metabolism, biosynthesis, and signaling of phytohormones, reprogrammed in a cell-type-specific manner. Our study provides a detailed spatiotemporal transcriptome of rice embryo and presents a previously undescribed methodology for exploring the roles of different embryonic cells in seed germination.

Case report: Successful management of primary hyalinizing clear cell carcinoma in nasopharynx: a report of 2 cases and system analysis.

Hyalinizing clear cell carcinomas (HCCCs) are infrequent, malignant tumors characterized by their low-grade nature. They typically originate from minor salivary glands. However, these tumors can potentially emerge in any location with minor salivary glands, including the nasopharynx. This report presents two cases of HCCC in females aged 61 and 72 years, with both tumors approximately 4 cm in size. In the first case, a 72-year-old female presented with recurrent bilateral epistaxis. Imaging studies revealed a nasopharyngeal mass, surgically excised, and histopathological analysis confirmed HCCC. Postoperatively, the patient received combined chemotherapy and radiotherapy, achieving a recurrence-free status 2.5 years later. The second case involves a 61-year-old female with a two-year history of bloody nasal discharge. Imaging studies identified a nasopharyngeal lesion, surgically removed, and histopathological examination confirmed HCCC. This patient underwent radiotherapy followed by combination chemotherapy with paclitaxel and carboplatin, displaying no signs of recurrence upon reevaluation after 10 months. These cases highlight the successful management of HCCC through a comprehensive, multimodal approach, integrating surgical intervention and adjuvant therapy. The favorable outcomes emphasize the significance of a thorough treatment strategy for HCCC in the nasopharynx, providing valuable insights for clinicians. Further studies are essential to enhance our understanding of this rare entity and refine treatment protocols for optimized patient outcomes.

Characterization of near-field temporal and spatial variations of pesticide residues using honeybee specimens as bio-sensing matrices.

This study was prompted by recent reports of the ubiquity of neonicotinoids (neonics) in environment and the likelihood of exposures and health hazards to non-target organisms. We aimed to quantify neonics levels in time- and location-match pollen and nectar samples foraged by honeybees (Apis mellifera) and characterized the temporal and spatial variations using a relative potency factor method to determine the total neonic levels, expressed as the imidacloprid-adjusted total neonics, IMIRPF (ng/g). Six pairs of pollen and nectar samples, a total of twelve samples, were collected from each of the thirty-two experimental hives during the active foraging months of March, April, and June and analyzed for eight neonics. We found 59% and 64% of pollen and nectar contained at least one neonic, respectively. Among those neonic-detected pollen and nectar samples, 45% and 77% of them contained more than one neonic, respectively. Imidacloprid and acetamiprid in pollen and clothianidin and thiamethoxam in nectar accounted for 60% and 83% detection, respectively. The highest 3-month average of IMIRPF in pollen (6.56 ng/g) and nectar (11.19 ng/g) were detected in a location with the predominant production of citrus fruit. The temporal and spatial variations of IMIRPF levels demonstrated the robustness of using paired pollen and nectar data as the bio-sensing matrices to facilitate the assessment of near-field exposure to total neonics and the delineation of risks.

PANX1-mediated ATP release confers FAM3A's suppression effects on hepatic gluconeogenesis and lipogenesis.

BACKGROUND: Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet ß cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed. METHODS: db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays. RESULTS: In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers. CONCLUSIONS: PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A's suppressive effects on hepatic gluconeogenesis and lipogenesis.

Integrated single-cell analysis defines the epigenetic basis of castration-resistant prostate luminal cells.

Understanding prostate response to castration and androgen receptor signaling inhibitors (ARSI) is critical to improving long-term prostate cancer (PCa) patient survival. Here, we use a multi-omics approach on 229,794 single cells to create a mouse single-cell reference atlas for interpreting mouse prostate biology and castration response. Our reference atlas refines single-cell annotations and provides a chromatin context, which, when coupled with mouse lineage tracing, demonstrates that castration-resistant luminal cells are distinct from the pre-existent urethra-proximal stem/progenitor cells. Molecular pathway analysis and therapeutic studies further implicate AP1 (JUN/FOS), WNT/ß-catenin, FOXQ1, NF-κB, and JAK/STAT pathways as major drivers of castration-resistant luminal populations with relevance to human PCa. Our datasets, which can be explored through an interactive portal (https://visportal.roswellpark.org/data/tang/), can aid in developing combination treatments with ARSI for advanced PCa patients.