Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

Endosome positioning coordinates spatially selective GPCR signaling.

G-protein-coupled receptors (GPCRs) can initiate unique functional responses depending on the subcellular site of activation. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have concentrated on the biochemical aspect of this regulation. Here we assess the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism. We devise a strategy to rapidly and selectively redistribute receptor-containing endosomes 'on command' in intact cells without perturbing their biochemical composition. Next, we present two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cyclic AMP (cAMP)-dependent transcriptional signaling with single-cell resolution. With these, we establish that disruption of native endosome positioning inhibits the initiation of the endosome-dependent transcriptional responses. Finally, we demonstrate a prominent mechanistic role of PDE-mediated cAMP hydrolysis and local protein kinase A activity in this process. Our study, therefore, illuminates a new mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially selective receptor signaling.

Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC.

The protein kinase C (PKC) family of serine and threonine kinases, consisting of three distinctly regulated subfamilies, has been established as critical for various cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs, is unclear. Here we present a sensitive excitation ratiometric C kinase activity reporter (ExRai-CKAR2) that enables the detection of minute changes (equivalent to 0.2% of maximum stimulation) in subcellular PKC activity. Using ExRai-CKAR2 with an enhanced diacylglycerol (DAG) biosensor, we uncover that G-protein-coupled receptor stimulation triggers sustained PKC activity at the endoplasmic reticulum and lysosomes, differentially mediated by Ca2+-sensitive conventional PKC and DAG-sensitive novel PKC, respectively. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or partitioning defective complexes, further enabled us to detect previously inaccessible endogenous atypical PKC activity in three-dimensional organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.

Post-implantation analysis of genomic variations in the progeny from developing fetus to birth.

The analysis of genomic variations in offspring after implantation has been infrequently studied. In this study, we aim to investigate the extent of de novo mutations in humans from developing fetus to birth. Using high-depth whole-genome sequencing, 443 parent-offspring trios were studied to compare the results of de novo mutations (DNMs) between different groups. The focus was on fetuses and newborns, with DNA samples obtained from the families' blood and the aspirated embryonic tissues subjected to deep sequencing. It was observed that the average number of total DNMs in the newborns group was 56.26 (54.17-58.35), which appeared to be lower than that the multifetal reduction group, which was 76.05 (69.70-82.40) (F = 2.42, P = 0.12). However, after adjusting for parental age and maternal pre-pregnancy body mass index (BMI), significant differences were found between the two groups. The analysis was further divided into single nucleotide variants (SNVs) and insertion/deletion of a small number of bases (indels), and it was discovered that the average number of de novo SNVs associated with the multifetal reduction group and the newborn group was 49.89 (45.59-54.20) and 51.09 (49.22-52.96), respectively. No significant differences were noted between the groups (F = 1.01, P = 0.32). However, a significant difference was observed for de novo indels, with a higher average number found in the multifetal reduction group compared to the newborn group (F = 194.17, P < 0.001). The average number of de novo indels among the multifetal reduction group and the newborn group was 26.26 (23.27-29.05) and 5.17 (4.82-5.52), respectively. To conclude, it has been observed that the quantity of de novo indels in the newborns experiences a significant decrease when compared to that in the aspirated embryonic tissues (7-9 weeks). This phenomenon is evident across all genomic regions, highlighting the adverse effects of de novo indels on the fetus and emphasizing the significance of embryonic implantation and intrauterine growth in human genetic selection mechanisms.

From phenotype to mechanism: Prenatal spectrum of NKAP mutation-related disorder and its pathogenesis inducing congenital heart disease.

NKAP mutations are associated with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD, MIM: #301039). Here, we elucidate the potential prenatal manifestation of NKAP mutation-associated disorder for the first time, alongside revealing the relationship between NKAP mutations and congenital heart defect (CHD) in the Chinese population. An NKAP mutation (NM_024528.4: c.988C>T, p.Arg330Cys) was identified in two foetuses presenting with CHD. Subsequent mechanistic exploration revealed a marked downregulation of NKAP transcription within HEK293T cells transfected with NKAP p.R330C. However, no significant change was observed at the protein level. Moreover, the mutation led to a dysregulation in the transcription of genes associated with cardiac morphogenesis, such as DHRS3, DNAH11 and JAG1. Additionally, our research determined that NKAP p.R330C affected Nkap protein intra-nuclear distribution, and binding with Hdac3. Summarily, our study strengthens NKAP mutations as a cause of CHD and prompts the reclassification of NKAP p.R330C as likely pathogenic, thereby establishing a prospective prenatal phenotypic spectrum that provides new insight into the prenatal diagnosis of CHD. Our findings also provide evidence of NKAP p.R330C pathogenicity and demonstrate the potential mechanism by which p.R330C dysregulates cardiac developmental gene transcription by altering Nkap intra-nuclear distribution and obstructing the interaction between Nkap and Hdac3, thereby leading to CHD.

FANCD2 as a ferroptosis-related target for recurrent implantation failure by integrated bioinformatics and Mendelian randomization analysis.

Despite advancements in assisted reproductive technology, recurrent implantation failure (RIF) remains a challenge. Endometrial factors, including ferroptosis and immunity, may contribute to this issue. This study integrated bioinformatics analysis and Mendelian randomization (MR) to investigate the expression and significance of DEFRGs in RIF. We intersected 484 ferroptosis-associated genes with 515 differentially expressed genes (DEGs) to identify key DEFRGs. Subsequent analyses included enrichment analysis, molecular subtype identification, machine learning model development for biomarker discovery, immune cell infiltration assessment, single-cell RNA sequencing, and MR to explore the causal relationships of selected genes with RIF. In this study, we identified 11 differentially expressed ferroptosis-related genes (DEFRGs) between RIF and healthy individuals. Cluster analysis revealed two distinct molecular subtypes with different immune profiles and DEFRG expressions. Machine learning models highlighted MUC1, GJA1 and FANCD2 as potential diagnostic biomarkers, with high accuracy in RIF prediction. Single-cell analysis further revealed the cellular localization and interactions of DEFRGs. MR suggested a protective effect of FANCD2 against RIF. Validation in RIF patients confirmed the differential expression of key DEFRGs, consistent with bioinformatics findings. This comprehensive study emphasize the significant role of DEFRGs in the pathogenesis of RIF, suggesting that modulating these genes could offer new avenues for treatment. The FANCD2 is a potential gene contributing to RIF pathogenesis through a non-classical ferroptosis-dependent pathway, providing a foundation for personalized therapeutic strategies in RIF management.

Signaling Microdomains in the Spotlight: Visualizing Compartmentalized Signaling Using Genetically Encoded Fluorescent Biosensors.

How cells muster a network of interlinking signaling pathways to faithfully convert diverse external cues to specific functional outcomes remains a central question in biology. Through their ability to convert dynamic biochemical activities to rapid and precise optical readouts, genetically encoded fluorescent biosensors have become instrumental in unraveling the molecular logic controlling the specificity of intracellular signaling. In this review, we discuss how the use of genetically encoded fluorescent biosensors to visualize dynamic signaling events within their native cellular context is elucidating the different strategies employed by cells to organize signaling activities into discrete compartments, or signaling microdomains, to ensure functional specificity.

Lenvatinib plus pembrolizumab for patients with previously treated, advanced, triple-negative breast cancer: Results from the triple-negative breast cancer cohort of the phase 2 LEAP-005 Study.

BACKGROUND: Novel treatments are needed for patients with advanced, triple-negative breast cancer (TNBC) that progresses or recurs after first-line treatment with chemotherapy. The authors report results from the TNBC cohort of the multicohort, open-label, single-arm, phase 2 LEAP-005 study of lenvatinib plus pembrolizumab in patients with advanced solid tumors (ClinicalTrials.gov identifier NCT03797326). METHODS: Eligible patients had metastatic or unresectable TNBC with disease progression after one or two lines of therapy. Patients received lenvatinib (20 mg daily) plus pembrolizumab (200 mg every 3 weeks; up to 35 cycles). The primary end points were the objective response rate according to Response Evaluation Criteria in Solid Tumors, version 1.1, and safety (adverse events graded by the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 4.0). Duration of response, progression-free survival, and overall survival were secondary end points. RESULTS: Thirty-one patients were enrolled. The objective response rate by investigator assessment was 23% (95% confidence interval [CI], 10%-41%). Overall, the objective response rate by blinded independent central review (BICR) was 32% (95% CI, 17%-51%); and, in patients who had programmed cell death ligand 1 combined positive scores ≥10 (n = 8) and <10 (n = 22), the objective response rate was 50% (95% CI, 16%-84%) and 27% (95% CI, 11%-50%), respectively. The median duration of response by BICR was 12.1 months (range, from 3.0+ to 37.9+ months). The median progression-free survival by BICR was 5.1 months (95% CI, 1.9-11.8 months) and the median overall survival was 11.4 months (95% CI, 4.1-21.7 months). Treatment-related adverse events occurred in 94% of patients (grade 3, 52%; grade 4, 0%). One patient died due to a treatment-related adverse event of subarachnoid hemorrhage. CONCLUSIONS: The combination of lenvatinib plus pembrolizumab demonstrated antitumor activity with a manageable safety profile in patients with previously treated, advanced TNBC.

Lenvatinib plus pembrolizumab for patients with previously treated advanced ovarian cancer: Results from the phase 2 multicohort LEAP-005 study.

OBJECTIVES: The phase 2, multicohort, open-label LEAP-005 study evaluated lenvatinib plus pembrolizumab in patients with previously treated advanced solid tumors. We report outcomes from the ovarian cancer cohort. METHODS: Eligible patients had metastatic/unresectable ovarian cancer and had received 3 previous lines of therapy. Patients received lenvatinib 20 mg/day plus pembrolizumab 200 mg every 3 weeks. Treatment continued until progression, unacceptable toxicity, or (for pembrolizumab) completion of 35 cycles. Primary endpoints were objective response rate (ORR) per RECIST version 1.1 and safety. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), and overall survival (OS). RESULTS: Thirty-one patients were enrolled. 39% had high grade serous ovarian cancer, 23% were platinum-sensitive, 55% were platinum-resistant, 23% were platinum-refractory, and 84% had tumors that had a PD-L1 combined positive (CPS) score ≥1. ORR (95% CI) was 26% (12%-45%) by investigator assessment and 35% (19%-55%) by blinded independent central review (BICR). Per BICR, median DOR was 9.2 (1.5+ to 37.8+) months. ORRs (95% CI) by BICR were 35% (9/26 patients; 17%-56%) for PD-L1 CPS ≥ 1 disease and 50% (2/4 patients; 7%-93%) for PD-L1 CPS < 1 disease. Median (95% CI) PFS by BICR and OS were 6.2 (4.0-8.5) months and 21.3 (11.7-32.3) months, respectively. Treatment-related AEs occurred in 94% of patients (grade 3-4, 77%). One patient died from treatment-related hypovolemic shock. CONCLUSIONS: Lenvatinib plus pembrolizumab demonstrated antitumor activity as fourth line therapy in patients with advanced ovarian cancer, and no unanticipated safety signals were identified. Responses were observed regardless of PD-L1 status.

Prevalence of homologous recombination biomarkers in multiple tumor types: an observational study.

Aim: To determine the prevalence of deleterious mutations in BRCA1 and BRCA2 and in 13 genes involved in homologous recombination repair (HRR), the prevalence of genomic loss of heterozygosity and the allelic and hereditary status of BRCA1, BRCA2 and other HRR gene mutations in multiple solid tumor types. Patients & methods: This was a retrospective observational study of patients with an advanced/metastatic diagnosis in one of 15 solid tumor types, who were identified in a real-world clinico-genomic database. Results: Tumor tissue samples from 9457 patients were analyzed, among which 4.7% had known or suspected deleterious BRCA1/2 mutations. The prevalence (range) of mutations in HRR genes was 13.6% (2.4%-26.0%) and genomic loss of heterozygosity ≥16% was 20.6% (2.6-34.4%) across all tumor types. Conclusion: The prevalence of mutations varied significantly depending on the type of tumor.

The integrity of the human genome is maintained via multiple pathways of DNA repair, one of the most important of which is homologous recombination repair (HRR), which uses a sister chromatid as a template for high-fidelity restoration of altered DNA sequences. This study aimed to determine the prevalence of deleterious mutations, i.e., changes in the genetic code that interfere with proper cellular function, in the breast cancer genes BRCA1 and BRCA2 and in 13 other genes involved in HRR in various types of solid tumors in patients with advanced or metastatic cancer. The researchers found that 4.7% of tumor samples had BRCA1/2 mutations, 13.6% had mutations in any of the HRR genes and 20.6% had genomic loss of heterozygosity (gLOH) of at least 16% i.e., loss of sections of chromosomes affecting 16% or more of the genome. BRCA1/2 mutations were most common in ovarian cancer (13.1%), prostate cancer (9.3%), breast cancer (8.2%) and pancreatic cancer (4.9%). Prevalence for mutations in HRR genes ranges from 2.4 to 26.0% and gLOH ≥16% ranged from 2.6 to 34.4% depending on the tumor type. In conclusion, the prevalence of mutations in the BRCA1/2 genes, HRR genes and gLOH ≥16% varied widely across 15 tumor types.

The yield of SNP microarray analysis for fetal ultrasound cardiac abnormalities.

BACKGROUND: Chromosomal microarray analysis (CMA) has emerged as a critical instrument in prenatal diagnostic procedures, notably in assessing congenital heart diseases (CHD). Nonetheless, current research focuses solely on CHD, overlooking the necessity for thorough comparative investigations encompassing fetuses with varied structural abnormalities or those without apparent structural anomalies. OBJECTIVE: This study sought to assess the relation of single nucleotide polymorphism-based chromosomal microarray analysis (SNP-based CMA) in identifying the underlying causes of fetal cardiac ultrasound abnormalities. METHODS: A total of 2092 pregnant women who underwent prenatal diagnosis from 2017 to 2022 were included in the study and divided into four groups based on the presence of ultrasound structural abnormalities and the specific type of abnormality. The results of the SNP-Array test conducted on amniotic fluid samples from these groups were analyzed. RESULTS: Findings from the study revealed that the non-isolated CHD group exhibited the highest incidence of aneuploidy, overall chromosomal abnormalities, and trisomy 18, demonstrating statistically significant differences from the other groups (p < 0.001). Regarding the distribution frequency of copy number variation (CNV) segment size, no statistically significant distinctions were observed between the isolated CHD group and the non-isolated CHD group (p > 0.05). The occurrence rates of 22q11.2 and 15q11.2 were also not statistically different between the isolated CHD group and the non-isolated congenital heart defect group (p > 0.05). CONCLUSION: SNP-based CMA enhances the capacity to detect abnormal CNVs in CHD fetuses, offering valuable insights for diagnosing chromosomal etiology and facilitating genetic counseling. This research contributes to the broader understanding of the utility of SNP-based CMA in the context of fetal cardiac ultrasound abnormalities.

Y chromosome polymorphisms contribute to an increased risk of non-obstructive azoospermia: a retrospective study of 32,055 Chinese men.

PURPOSE: To investigate the prevalence of Y chromosome polymorphisms in Chinese men and analyze their associations with male infertility and female adverse pregnancy outcomes. METHODS: The clinical data of 32,055 Chinese men who underwent karyotype analysis from October 2014 to September 2019 were collected. Fisher's exact test, chi-square test, or Kruskal-Wallis test was used to analyze the effects of Y chromosome polymorphism on semen parameters, azoospermia factor (AZF) microdeletions, and female adverse pregnancy outcomes. RESULTS: The incidence of Y chromosome polymorphic variants was 1.19% (381/32,055) in Chinese men. The incidence of non-obstructive azoospermia (NOA) was significantly higher in men with the Yqh- variant than that in men with normal karyotype and other Y chromosome polymorphic variants (p < 0.050). The incidence of AZF microdeletions was significantly different among the normal karyotype and different Y chromosome polymorphic variant groups (p < 0.001). The detection rate of AZF microdeletions was 28.92% (24/83) in the Yqh- group and 2.50% (3/120) in the Y ≤ 21 group. The AZFb + c region was the most common AZF microdeletion (78.57%, 22/28), followed by AZFc microdeletion (7.14%,2/28) in NOA patients with Yqh- variants. There was no significant difference in the distribution of female adverse pregnancy outcomes among the normal karyotype and different Y chromosome polymorphic variant groups (p = 0.528). CONCLUSIONS: Patients with 46,XYqh- variant have a higher incidence of NOA and AZF microdeletions than patients with normal karyotype and other Y chromosome polymorphic variants. Y chromosome polymorphic variants do not affect female adverse pregnancy outcomes.

Clinical Study on the Thoracodorsal Artery Perforator Flap in Breast-Conserving Reconstruction of T2 Breast Cancer.

BACKGROUND: Breast-conserving surgery combined with oncoplastic breast surgery has become the standard surgical treatment for early breast cancer. OBJECTIVE: The purpose of this study was to investigate the safety and efficacy of the thoracodorsal artery perforator flap (TDAPF) in breast-conserving reconstruction of T2 breast cancer. METHODS: Thirty patients with T2 breast cancer admitted to our hospital from January 2019 to December 2020 were enrolled to receive pedicled TDAPF for repairing breast defects after breast-conserving surgery. Intraoperative conditions, postoperative complications, and shape satisfaction after breast reconstruction were recorded. RESULTS: The operation was successfully completed in all 30 patients, with an operation time of 177.77 ± 24.39 min, bleeding of 44.17 ± 7.67 mL, and length of hospital stay of 5.23 ± .97 d. There was no deformity or seroma at the donor site. Breast shape recovered well after operation. After operation, one patient had fat liquefaction in the recipient site, which healed well after wound treatment. The incidence of postoperative complications was 3.33%. Postoperative follow-up lasted 16-28 months, with a median of 22 months. The Breast-Q score for breast satisfaction was 61.83 ± 12.87 at 6 months after operation, compared to 62.07 ± 11.78 before operation (P > .05). CONCLUSIONS: TDAPF, featuring a high survival rate, moderate flap area, fewer postoperative complications, and high satisfaction with breast shape after operation. For east asian women with moderate breast size, TDAPF is a safe, effective choice for repairing defects in breast-conserving surgery for T2 breast cancer.

Research progress on the regulation of oxidative stress by phenolics: the role of gut microbiota and Nrf2 signaling pathway.

In recent years, the increase in high-calorie diets and sedentary lifestyles has made obesity a global public health problem. An unbalanced diet promotes the production of proinflammatory cytokines and causes redox imbalance in the body. Phenolics have potent antioxidant activity and cytoprotective ability. They can scavenge free radicals and reactive oxygen species, and enhance the activity of antioxidant enzymes, thus combating the body's oxidative stress. They can also improve the body's inflammatory response, enhance the enzyme activity of lipid metabolism, and reduce the contents of cholesterol and triglyceride. Most phenolics are biotransformed and absorbed into the blood after the action by gut microbiota; these metabolites then undergo phase I and II metabolism and regulate oxidative stress by scavenging free radicals and increasing expression of antioxidant enzymes. Phenolics induce the expression of genes encoding antioxidant enzymes and phase II detoxification enzymes by stimulating Nrf2 to enter the nucleus and bind to the antioxidant response element after uncoupling from Keap1, thereby promoting the production of antioxidant enzymes and phase II detoxification enzymes. The absorption rate of phenolics in the small intestine is extremely low. Most phenolics reach the colon, where they interact with the microbiota and undergo a series of metabolism. Their metabolites will reach the liver via the portal vein and undergo conjugation reactions. Subsequently, the metabolites reach the whole body to exert biological activity by traveling with the systemic circulation. Phenolics can promote the growth of probiotics, reduce the ratio of Firmicutes/Bacteroidetes (F/B), and improve intestinal microecological imbalance. This paper reviews the nutritional value, bioactivity, and antioxidant mechanism of phenolics in the body, aiming to provide a scientific basis for the development and utilization of natural antioxidants and provide a reference for elucidating the mechanism of action of phenolics for regulating oxidative stress in the body. © 2023 Society of Chemical Industry.

[Whole exome sequencing analysis of 37 fetuses with cardiac abnormalities].

OBJECTIVE: To explore the genetic etiology of fetuses with congenital heart disease (CHD) through whole exome sequencing (WES). METHODS: Thirty seven fetuses identified with CHD by prenatal ultrasonography but with negative results by chromosomal microarray analysis (CMA) at Jinhua Maternal and Child Health Care Hospital from January 2020 to June 2022 were selected as the study subjects, for whom WES was carried out. RESULTS: WES and Sanger sequencing had detected 6 pathogenic or likely pathogenic variants, and 6 variants with unknown clinical significance. The variants had involved 15 loci within 11 genes, in addition with one copy number variation. CONCLUSION: WES can increase the detection rate for genetic abnormalities among fetuses with CHD, which can facilitate the prenatal diagnosis, evaluation of prognosis and genetic counseling for the couples.

Efficacy and safety of pembrolizumab monotherapy in patients with advanced thyroid cancer in the phase 2 KEYNOTE-158 study.

BACKGROUND: The authors report results from the thyroid carcinoma cohort of the multicohort phase 2 KEYNOTE-158 study (NCT02628067), which evaluated pembrolizumab monotherapy in patients with previously treated cancers. METHODS: Eligible patients had histologically and/or cytologically confirmed papillary or follicular thyroid carcinoma, failure of or intolerance to prior therapy, and measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Patients received pembrolizumab (200 mg) every 3 weeks for up to 35 cycles. The primary end point was objective response rate (ORR) per RECIST v1.1 by independent central review. RESULTS: A total of 103 patients were enrolled and received pembrolizumab. Median duration from first dose to data cutoff (October 5, 2020) was 49.4 (range, 43.9-54.9) months. ORR was 6.8% (95% confidence interval [CI], 2.8%-13.5%), and median duration of response was 18.4 (range, 4.2-47.2+) months. ORR was 8.7% (95% CI, 2.4%-20.8%) among patients with programmed cell death ligand 1 (PD-L1) combined positive score (CPS) ≥1 (n = 46) and 5.7% (95% CI, 1.2%-15.7%) among patients with PD-L1 CPS <1 (n = 53). Median overall survival and progression-free survival were 34.5 (95% CI, 21.2 to not reached) and 4.2 (95% CI, 3.9-6.2) months, respectively. Treatment-related adverse events occurred in 69.9% of patients (grade 3-5, 14.6%). CONCLUSIONS: Pembrolizumab demonstrated manageable toxicity and durable antitumor activity in a small subset of patients with advanced thyroid cancer. These results provide evidence of modest antitumor activity in this setting regardless of tumor PD-L1 expression. Future studies evaluating immune checkpoint inhibitors in patients with differentiated thyroid cancer should focus on biomarker-driven patient selection or combination of immune checkpoint inhibitors with other agents, in order to achieve higher response rates than observed in this study.

Estrogen-sensitive activation of SGK1 induces M2 macrophages with anti-inflammatory properties and a Th2 response at the maternal-fetal interface.

BACKGROUND: Decidual macrophages participate in immune regulation at the maternal-fetal interface. Abnormal M1/M2 polarization of decidual macrophages might predispose immune maladaptation in recurrent pregnancy loss (RPL). However, the mechanism of decidual macrophage polarization is unclear. We explored the role of Estradiol (E2)-sensitive serum-glucocorticoid regulated kinase (SGK) 1 in promoting macrophage polarization and suppressing inflammation at the maternal-fetal interface. METHODS: We assessed serum levels of E2 and progesterone during first trimester of pregnancy in women with or without threatened miscarriages (ended in live birth, n = 448; or early miscarriages, n = 68). For detection of SGK1 in decidual macrophages, we performed immunofluorescence labeling and western blot analysis applying decidual samples from RPL (n = 93) and early normal pregnancy (n = 66). Human monocytic THP-1 cells were differentiated into macrophages and treated with Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS), E2, inhibitors or siRNA for in vitro analysis. Flow cytometry analysis were conducted to detect macrophages polarization. We also applied ovariectomized (OVX) mice with hormones exploring the mechanisms underlying the regulation of SGK1 activation by E2 in the decidual macrophages in vivo. RESULTS: SGK1 expression down regulation in the decidual macrophages of RPL was consistent with the lower concentration and slower increment of serum E2 from 4 to 12 weeks of gestation seen in these compromised pregnancies. LPS reduced SGK1 activities, but induced the pro-inflammatory M1 phenotype of THP-1 monocyte-derived macrophages and T helper (Th) 1 cytokines that favored pregnancy loss. E2 pretreatment promoted SGK1 activation in the decidual macrophages of OVX mice in vivo. E2 pretreatment amplified SGK1 activation in TLR4-stimulated THP-1 macrophages in vitro through the estrogen receptor beta (ERß) and PI3K pathway. E2-sensitive activation of SGK1 increased M2 macrophages and Th2 immune responses, which were beneficial to successful pregnancy, by inducing ARG1 and IRF4 transcription, which are implicated in normal pregnancy. The experiments on OVX mice have shown that pharmacological inhibition of E2 promoted nuclear translocation of NF-κB in the decidual macrophages. Further more, pharmacological inhibition or knockdown of SGK1 in TLR4-stimulated THP-1 macrophages activated NF-κB by promoting its nuclear translocation, leading to increased secretion of pro-inflammatory cytokines involved in pregnancy loss. CONCLUSION: Our findings highlighted the immunomodulatory roles of E2-activated SGK1 in Th2 immune responses by priming anti-inflammatory M2 macrophages at the maternal-fetal interface, resulting in a balanced immune microenvironment during pregnancy. Our results suggest new perspectives on future preventative strategies for RPL.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

MicroRNA: Crucial modulator in purinergic signalling involved diseases.

Both microRNAs (miRNAs) and purinergic signalling are widely and respectively expressed in various tissues of different organisms and play vital roles in a variety of physiological and pathological processes. Here, we reviewed the current publications contributed to the relationship of miRNAs and purinergic signalling in cardiovascular diseases, gastrointestinal diseases, neurological diseases, and ophthalmic diseases. We tried to decode the miRNAs-purinergic signalling network of purinergic signalling involved diseases. The evidence indicated that more than 30 miRNAs (miR-22, miR-30, miR-146, miR-150, miR-155, miR-187, etc.) directly or indirectly modulate P1 receptors (A1, A2A, A2B, A3), P2 receptors (P2X1, P2X3, P2X4, P2X7, P2Y2, P2Y6, P2Y12), and ecto-enzymes (CD39, CD73, ADA2); P2X7 and CD73 could be modulated by multiple miRNAs (P2X7: miR-21, miR-22, miR-30, miR-135a, miR-150, miR-186, miR-187, miR-216b; CD73: miR-141, miR-101, miR-193b, miR-340, miR-187, miR-30, miR-422a); miR-187 would be the common miRNA to modulate P2X7 and CD73.

Mercuric chloride induced brain toxicity in mice: The protective effects of puerarin-loaded PLGA nanoparticles.

Mercury is a toxic, environmentally heavy metal that can cause severe damage to all organs, including the nervous system. The functions of puerarin include antioxidant, anti-inflammatory, nerve cell repair, regulation of autophagy, and so forth. But because of the limited oral absorption of puerarin, it affects the protective effect on brain tissue. The nano-encapsulation of Pue can improve its limitation. Therefore, this study investigated the protective effect of Pue drug-loaded PLGA nanoparticles (Pue-PLGA-nps) on brain injury induced by mercuric chloride (HgCl2 ) in mice. The mice were divided into normal saline (NS) group, HgCl2 (4 mg/kg) group, Pue-PLGA-nps (50 mg/kg) group, HgCl2 + Pue (4 mg/kg + 30 mg/kg) group, and HgCl2 + Pue-PLGA-nps (4 mg/kg + 50 mg/kg) group. After 28 days of treatment, the mice were observed for behavioral changes, antioxidant capacity, autophagy and inflammatory response, and mercury levels in the brain, blood, and urine were measured. The results showed that HgCl2 toxicity caused learning and memory dysfunction in mice, increased mercury content in brain and blood, and increased serum levels of interleukin (IL-6), IL-1ß, and tumor necrosis factor-α in the mice. HgCl2 exposure decreased the activity of T-AOC, superoxide dismutase, and glutathione peroxidase, and increased the expression of malondialdehyde in the brain of mice. Moreover, the expression levels of TRIM32, toll-like receptor 4 (TLR4), and LC3 proteins were upregulated. Both Pue and Pue-PLGA-nps interventions mitigated the changes caused by HgCl2 exposure, and Pue-PLGA-nps further enhanced this effect. Our results suggest that Pue-PLGA-nps can ameliorate HgCl2 -induced brain injury and reduce Hg accumulation, which is associated with inhibition of oxidative stress, inflammatory response, and TLR4/TRIM32/LC3 signaling pathway.