Development of an ubiquitin-proteasome system signature for predicting prognosis and providing therapeutic guidance for patients with triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a pathological subtype with a high mortality, and the development of inhibitors in the ubiquitin-proteasome system (UPS) component could be a novel therapeutic tool. METHODS: Triple-negative breast cancer data were obtained from The Cancer Genome Atlas (TCGA), and subtype analysis was performed by consistent clustering analysis to identify molecular subtypes of TNBC according to UPS characteristics. Differential analysis, COX and least absolute shrinkage and selection operator (LASSO) COX regression analyses were performed to select genes associated with overall survival in TNBC. The final prognostic model (UPS score) was determined using the LASSO COX model. The model performance was assessed using receiver operating characteristic (ROC) curves and survival curves. In addition, the results of the UPS score on analyzing the abundance of immune cell infiltration and immunotherapy were explored. Finally, we developed a nomogram for TNBC survival prediction. RESULTS: Two UPS subtypes (UPSMS1 and UPSMS2) showing significant survival differences were classified. COX regression analysis on differentially expressed genes in UPSMS1 and UPSMS2 filtered five genes that affected overall survival. Based on the regression coefficients and expression data of the five genes, we built a prognostic assessment system (UPS score). The UPS score showed consistent prognostic and therapeutic guidance values. Finally, the ROC curve of the nomogram and UPS score showed the highest predictive efficacy compared with traditional clinical prognostic indicators. CONCLUSION: The UPS score represented a promising prognostic tool to predict overall survival and immune status and guide personalized treatment selection in TNBC patients, and this study may provide a more practical alternative for clinical monitoring and management of TNBC.

Complete degradation of di-n-butyl phthalate by Glutamicibacter sp. strain 0426 with a novel pathway.

Di-n-butyl phthalate (DBP) is widely used as plasticizer that has potential carcinogenic, teratogenic, and endocrine effects. In the present study, an efficient DBP-degrading bacterial strain 0426 was isolated and identified as a Glutamicibacter sp. strain 0426. It can utilize DBP as the sole source of carbon and energy and completely degraded 300 mg/L of DBP within 12 h. The optimal conditions (pH 6.9 and 31.7 °C) for DBP degradation were determined by response surface methodology and DBP degradation well fitted with the first-order kinetics. Bioaugmentation of contaminated soil with strain 0426 enhanced DBP (1 mg/g soil) degradation, indicating the application potential of strain 0426 for environment DBP removal. Strain 0426 harbors a distinctive DBP hydrolysis mechanism with two parallel benzoate metabolic pathways, which may account for the remarkable performance of DBP degradation. Sequences alignment has shown that an alpha/beta fold hydrolase (WP_083586847.1) contained a conserved catalytic triad and pentapeptide motif (GX1SX2G), of which function is similar to phthalic acid ester (PAEs) hydrolases and lipases that can efficiently catalyze hydrolysis of water-insoluble substrates. Furthermore, phthalic acid was converted to benzoate by decarboxylation, which entered into two different pathways: one is the protocatechuic acid pathway under the role of pca cluster, and the other is the catechol pathway. This study demonstrates a novel DBP degradation pathway, which broadens our understanding of the mechanisms of PAE biodegradation.

Synthetic Nuclease-Producing Microbiome Achieves Efficient Removal of Extracellular Antibiotic Resistance Genes from Wastewater Effluent.

Antibiotic resistance gene (ARG) transmission poses significant threats to human health. The effluent of wastewater treatment plants is demonstrated as a hotspot source of ARGs released into the environment. In this study, a synthetic microbiome containing nuclease-producing Deinococcus radiodurans was constructed to remove extracellular ARGs. Results of quantitative polymerase chain reaction (qPCR) showed significant reduction in plasmid RP4-associated ARGs (by more than 3 orders of magnitude) and reduction of indigenous ARG sul1 and mobile genetic element (MGE) intl1 (by more than 1 order of magnitude) in the synthetic microbiome compared to the control without D. radiodurans. Metagenomic analysis revealed a decrease in ARG and MGE diversity in extracellular DNA (eDNA) of the treated group. Notably, whereas eight antibiotic-resistant plasmids with mobility risk were detected in the control, only one was detected in the synthetic microbiome. The abundance of the nuclease encoding gene exeM, quantified by qPCR, indicated its enrichment in the synthetic microbiome, which ensures stable eDNA degradation even when D. radiodurans decreased. Moreover, intracellular ARGs and MGEs and pathogenic ARG hosts in the river receiving treated effluent were lower than those in the river receiving untreated effluent. Overall, this study presents a new approach for removing extracellular ARGs and further reducing the risk of ARG transmission in receiving rivers.

Distinct clinical and somatic mutational features of breast tumors with high-, low-, or non-expressing human epidermal growth factor receptor 2 status.

BACKGROUND: HER2-low breast cancers were reported to have distinct clinicopathological characteristics from HER2-zero; however, the difference in their genetic features remains unclear. This study investigated the clinical and molecular features of breast tumors according to HER2 status. METHODS: We analyzed the clinicopathological and genomic data of 523 Chinese women with breast cancer. Genomic data was generated by targeted next-generation sequencing (NGS) of breast tumor samples using a commercial 520 gene panel. The cohort was stratified according to HER2 status as HER2-zero (n = 90), HER2-low (n = 231), and HER2-positive (n = 202) according to their immunohistochemistry and fluorescence in situ hybridization results. RESULTS: HER2-low breast tumors were enriched with hormone receptor-positive tumors, and who had lower Ki67 expression levels. Genes were differentially mutated across HER2 subgroups. HER2-low tumors had significantly more mutations involved in PI3K-Akt signaling than HER2-positive (p < 0.001) and HER2-zero breast tumors (p < 0.01). HER2-zero tumors had more mutations in checkpoint factors (p < 0.01), Fanconi anemia (p < 0.05), and p53 signaling and cell cycle pathway (p < 0.05) compared to HER2-low breast tumors. Compared with HER2-zero tumors, HER2-low tumors had significantly lower pathological complete response rates after neoadjuvant therapy (15.9% vs. 37.5%, p = 0.042) and proportion of relapsed/progressed patients across follow-up time points (p = 0.031), but had comparable disease-free survival (p = 0.271). CONCLUSION: Our results demonstrate the distinct clinical and molecular features and clinical outcomes of HER2-low breast tumors.

Adagrasib, a KRAS G12C inhibitor, reverses the multidrug resistance mediated by ABCB1 in vitro and in vivo.

BACKGROUND: Multidrug resistance (MDR) is a complex phenomenon that frequently leads to chemotherapy failure during cancer treatment. The overexpression of ATP-binding cassette (ABC) transporters represents the major mechanism contributing to MDR. To date, no effective MDR modulator has been applied in clinic. Adagrasib (MRTX849), a specific inhibitor targeting KRAS G12C mutant, is currently under investigation in clinical trials for the treatment of non-small cell lung cancer (NSCLC). This study focused on investigating the circumvention of MDR by MRTX849. METHODS: The cytotoxicity and MDR reversal effect of MRTX849 were assessed by MTT assay. Drug accumulation and drug efflux were evaluated by flow cytometry. The MDR reversal by MRTX849 in vivo was investigated in two ABCB1-overexpressing tumor xenograft models in nude mice. The interaction between MRTX849 and ABCB1 substrate binding sites was studied by the [125I]-IAAP-photoaffinity labeling assay. The vanadate-sensitive ATPase assay was performed to identify whether MRTX849 would change ABCB1 ATPase activity. The effect of MRTX849 on expression of ABCB1 and PI3K/AKT signaling molecules was examined by flow cytometry, Western blot and Quantitative Real-time PCR analyses. RESULTS: MRTX849 was shown to enhance the anticancer efficacy of ABCB1 substrate drugs in the transporter-overexpressing cells both in vitro and in vivo. The MDR reversal effect was specific against ABCB1 because no similar effect was observed in the parental sensitive cells or in ABCG2-mediated MDR cells. Mechanistically, MRTX849 increased the cellular accumulation of ABCB1 substrates including doxorubicin (Dox) and rhodamine 123 (Rho123) in ABCB1-overexpressing MDR cells by suppressing ABCB1 efflux activity. Additionally, MRTX849 stimulated ABCB1 ATPase activity and competed with [125I]-IAAP for photolabeling of ABCB1 in a concentration-dependent manner. However, MRTX849 did not alter ABCB1 expression or phosphorylation of AKT/ERK at the effective MDR reversal drug concentrations. CONCLUSIONS: In summary, MRTX849 was found to overcome ABCB1-mediated MDR both in vitro and in vivo by specifically attenuating ABCB1 efflux activity in drug-resistant cancer cells. Further studies are warranted to translate the combination of MRTX849 and conventional chemotherapy to clinical application for circumvention of MDR. Video Abstract.

A Carotenoid- and Nuclease-Producing Bacterium Can Mitigate Enterococcus faecalis Transformation by Antibiotic Resistance Genes.

Dissemination of antibiotic resistance genes (ARGs) through natural transformation is facilitated by factors that stabilize extracellular DNA (eDNA) and that induce reactive oxygen species (ROS) that permeabilize receptor cells and upregulate transformation competence genes. In this study, we demonstrate that Deinococcus radiodurans can mitigate this ARG dissemination pathway by removing both eDNA and ROS that make recipient cells more vulnerable to transformation. We used plasmid RP4 as source of extracellular ARGs (tetA, aphA, and blaTEM-2) and the opportunistic pathogen Enterococcus faecalis as receptor. The presence of D. radiodurans significantly reduced the transformation frequency from 2.5 ± 0.7 × 10-6 to 7.4 ± 1.4 × 10-7 (p < 0.05). Based on quantification of intracellular ROS accumulation and superoxide dismutase (SOD) activity, and quantitative polymerase chain reaction (qPCR) and transcriptomic analyses, we propose two mechanisms by which D. radiodurans mitigates E. faecalis transformation by ARGs: (a) residual antibiotics induce D. radiodurans to synthesize liposoluble carotenoids that scavenge ROS and thus mitigate the susceptibility of E. faecalis for eDNA uptake, and (b) eDNA induces D. radiodurans to synthesize extracellular nucleases that degrade eARGs. This mechanistic insight informs biological strategies (including bioaugmentation) to curtail the spread of ARGs through transformation.

Biological Mitigation of Antibiotic Resistance Gene Dissemination by Antioxidant-Producing Microorganisms in Activated Sludge Systems.

Antibiotic resistance is the principal mechanism of an evergrowing bacterial threat. Antibiotic residues in the environment are a major contributor to the spread of antibiotic resistance genes (ARGs). Subinhibitory concentrations of antibiotics cause bacteria to produce reactive oxygen species (ROS), which can lead to mutagenesis and horizontal gene transfer (HGT) of ARGs; however, little is known about the mitigation of ARG dissemination through ROS removal by antioxidants. In this study, we examine how antioxidant-producing microorganisms inoculated in replicate activated sludge systems can biologically mitigate the dissemination of ARGs. Through quantitative polymerase chain reaction (qPCR), we showed that antioxidant-producing microorganisms could decrease the persistence of the RP4 plasmid and alleviate enrichment of ARGs (sul1) and class 1 integrons (intl1). Metagenomic sequencing identified the most diverse resistome and the most mutated Escherichia coli ARGs in the reactor that contained antibiotics but no antioxidant-producing microorganisms, suggesting that antioxidant-producing microorganisms mitigated ARG enrichment and mutation. Host classification revealed that antioxidant-producing microorganisms decreased the diversity of ARG hosts by shaping the microbial community through competition and functional pathway changes. Conjugative experiments demonstrated that conjugative transfer of ARGs could be mitigated by coculture with antioxidant-producing microorganisms. Overall, this is a novel study that shows how ARG enrichment and HGT can be mitigated through bioaugmentation with antioxidant-producing microorganisms.

Comprehensive analysis of autophagy-related prognostic genes in breast cancer.

Accumulating evidence revealed that autophagy played vital roles in breast cancer (BC) progression. Thus, the aim of this study was to investigate the prognostic value of autophagy-related genes (ARGs) and develop a ARG-based model to evaluate 5-year overall survival (OS) in BC patients. We acquired ARG expression profiling in a large BC cohort (N = 1007) from The Cancer Genome Atlas (TCGA) database. The correlation between ARGs and OS was confirmed by the LASSO and Cox regression analyses. A predictive model was established based on independent prognostic variables. Thus, time-dependent receiver operating curve (ROC), calibration plot, decision curve and subgroup analysis were conducted to determine the predictive performance of ARG-based model. Four ARGs (ATG4A, IFNG, NRG1 and SERPINA1) were identified using the LASSO and multivariate Cox regression analyses. A ARG-based model was constructed based on the four ARGs and two clinicopathological risk factors (age and TNM stage), dividing patients into high-risk and low-risk groups. The 5-year OS of patients in the low-risk group was higher than that in the high-risk group (P < 0.0001). Time-dependent ROC at 5 years indicated that the four ARG-based tool had better prognostic accuracy than TNM stage in the training cohort (AUC: 0.731 vs 0.640, P < 0.01) and validation cohort (AUC: 0.804 vs 0.671, P < 0.01). The mutation frequencies of the four ARGs (ATG4A, IFNG, NRG1 and SERPINA1) were 0.9%, 2.8%, 8% and 1.3%, respectively. We built and verified a novel four ARG-based nomogram, a credible approach to predict 5-year OS in BC, which can assist oncologists in determining effective therapeutic strategies.

Comparative genomics analysis of c-di-GMP metabolism and regulation in Microcystis aeruginosa.

BACKGROUND: Cyanobacteria are of special concern because they proliferate in eutrophic water bodies worldwide and affect water quality. As an ancient photosynthetic microorganism, cyanobacteria can survive in ecologically diverse habitats because of their capacity to rapidly respond to environmental changes through a web of complex signaling networks, including using second messengers to regulate physiology or metabolism. A ubiquitous second messenger, bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has been found to regulate essential behaviors in a few cyanobacteria but not Microcystis, which are the most dominant species in cyanobacterial blooms. In this study, comparative genomics analysis was performed to explore the genomic basis of c-di-GMP signaling in Microcystis aeruginosa. RESULTS: Proteins involved in c-di-GMP metabolism and regulation, such as diguanylate cyclases, phosphodiesterases, and PilZ-containing proteins, were encoded in M. aeruginosa genomes. However, the number of identified protein domains involved in c-di-GMP signaling was not proportional to the size of M. aeruginosa genomes (4.97 Mb in average). Pan-genome analysis showed that genes involved in c-di-GMP metabolism and regulation are conservative in M. aeruginosa strains. Phylogenetic analysis showed good congruence between the two types of phylogenetic trees based on 31 highly conserved protein-coding genes and sensor domain-coding genes. Propensity for gene loss analysis revealed that most of genes involved in c-di-GMP signaling are stable in M. aeruginosa strains. Moreover, bioinformatics and structure analysis of c-di-GMP signal-related GGDEF and EAL domains revealed that they all possess essential conserved amino acid residues that bind the substrate. In addition, it was also found that all selected M. aeruginosa genomes encode PilZ domain containing proteins. CONCLUSIONS: Comparative genomics analysis of c-di-GMP metabolism and regulation in M. aeruginosa strains helped elucidating the genetic basis of c-di-GMP signaling pathways in M. aeruginosa. Knowledge of c-di-GMP metabolism and relevant signal regulatory processes in cyanobacteria can enhance our understanding of their adaptability to various environments and bloom-forming mechanism.

Genetic mutation profile of Chinese HER2-positive breast cancers and genetic predictors of responses to Neoadjuvant anti-HER2 therapy.

PURPOSE: Despite the therapeutic success of existing HER2-targeted therapies, tumors respond quite differently to them. This study aimed at figuring out genetic mutation profile of Chinese HER2-positive patients and investigating predictive factors of neoadjuvant anti-HER2 responses. METHODS: We employed two cohorts. The first cohort was comprised of 181 HER2-positive patients treated at Guangdong Provincial People's Hospital from 2012 to 2018. The second cohort included 40 patients from the first cohort who underwent HER2-targeted neoadjuvant chemotherapy. Genetic mutations were characterized using next-generation sequencing. We employed the most commonly used definition of pathological complete response (pCR)-eradication of tumor from both breast and lymph nodes (ypT0/is ypN0). RESULTS: In Chinese HER2-positive breast cancer patients, TP53 (74.6%), CDK12 (64.6%) and PIK3CA (46.4%) have the highest mutation frequencies. In cohort 2, significant differences were found between pCR and non-pCR groups in terms of the initial Ki67 status, TP53 missense mutations, TP53 LOF mutations, PIK3CA mutations and ROS1 mutations (p = 0.028, 0.019, 0.005, 0.013, 0.049, respectively). Furthermore, TP53 LOF mutations and initial Ki67 status (OR 7.086, 95% CI 1.366-36.749, p = 0.020 and OR 6.007, 95% CI 1.120-32.210, p = 0.036, respectively) were found to be predictive of pCR status. CONCLUSION: TP53 LOF mutations and initial Ki67 status in HER2-positive breast cancer are predictive of pCR status after HER2-targeted NACT.

Genetic Bioaugmentation of Activated Sludge with Dioxin-Catabolic Plasmids Harbored by Rhodococcus sp. Strain p52.

Horizontal transfer of catabolic plasmids is used in genetic bioaugmentation for environmental pollutant remediation. In this study, we examined the effectiveness of genetic bioaugmentation with dioxin-catabolic plasmids harbored by Rhodococcus sp. strain p52 in laboratory-scale sequencing batch reactors (SBRs). During 100 days of operation, bioaugmentation decreased the dibenzofuran content (120 mg L-1) in the synthetic wastewater by 32.6%-100% of that in the nonbioaugmented SBR. Additionally, dibenzofuran was removed to undetectable levels in the bioaugmented SBR, in contrast, 46.8 ± 4.1% of that in the influent remained in the nonbioaugmented SBR after 96 days. Moreover, transconjugants harboring pDF01 and pDF02 were isolated from the bioaugmented SBR after 2 days, and their abilities to degrade dibenzofuran were confirmed. After 80 days, the copy numbers of strain p52 decreased by 3 orders of magnitude and accounted for 0.05 ± 0.01% of the total bacteria, while transconjugants were present at around 106 copies mL-1 sludge and accounted for 8.2 ± 0.3% of the total bacteria. Evaluation of the bacterial community profile of sludge by high-throughput 16S rRNA gene sequencing revealed that genetic bioaugmentation led to a bacterial community with an even distribution of genera in the SBR. This study demonstrates the promise of genetic bioaugmentation with catabolic plasmids for dioxins remediation.

Presence of circulating tumor cells is associated with metabolic-related variables in postoperative patients with early-stage breast cancer.

OBJECTIVE: Although circulating tumor cells (CTCs) have been well-established as promising prognostic biomarkers in both early breast cancer and metastatic settings, little is known regarding the prognostic relevance of CTCs in the long-term postoperative monitoring of patients with non-metastatic breast cancer (non-MBC). In this study, we investigated the associations of CTCs with clinicopathological features and metabolic-related variables, such as obesity and hyperglycemia. METHODS: In this retrospective study, we recruited 264 patients with postoperative stage I-III breast cancer at Guangdong General Hospital from January 2009 to December 2015. The prevalence and number of CTCs were assessed using the CellSearch System at a median time of 19.0 months [interquartile range (IQR), 7.8-33.0] after surgery. The CTC assay results were correlated with the clinicopathological features and metabolic-related variables. A multivariate logistic regression analysis was performed to further determine the independent predictors of CTCs. RESULTS: CTCs were detected in 10.6% of all patients. The positive rate of CTCs in patients with infiltrating ductal carcinoma was lower than that in patients with other pathological types (9.0% vs. 28.6%, P=0.020). More importantly, the presence of CTCs was correlated with blood glucose level (P=0.015) and high-density lipoprotein level (P=0.030). The multivariate logistic regression analysis showed that the pathological type [odds ratio (OR): 1.757, 95% CI: 1.021-3.023; P=0.042] and blood glucose level (OR: 1.218, 95% CI: 1.014-1.465; P=0.035) were independent predictors of the presence of CTCs. CONCLUSIONS: This study revealed potential associations between CTCs and metabolic-related factors in Chinese patients with non-MBC and supports the hypothesis that metabolic dysfunction in breast cancer patients might influence the biological activity of metastatic breast cancer, leading to a higher prevalence of CTCs.

Simultaneous antibiotic removal and mitigation of resistance induction by manganese bio-oxidation process.

Microbial degradation to remove residual antibiotics in wastewater is of growing interest. However, biological treatment of antibiotics may cause resistance dissemination by mutations and horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). In this study, a Mn(Ⅱ)-oxidizing bacterium (MnOB), Pseudomonas aeruginosa MQ2, simultaneously degraded antibiotics, decreased HGT, and mitigated antibiotic resistance mutation. Intracellular Mn(II) levels increased during manganese oxidation, and biogenic manganese oxides (BioMnOx, including Mn(II), Mn(III) and Mn(IV)) tightly coated the cell surface. Mn(II) bio-oxidation mitigated antibiotic resistance acquisition from an E. coli ARG donor and mitigated antibiotic resistance inducement by decreasing conjugative transfer and mutation, respectively. BioMnOx also oxidized ciprofloxacin (1 mg/L) and tetracycline (5 mg/L), respectively removing 93% and 96% within 24 h. Transcriptomic analysis revealed that two new multicopper oxidase and one peroxidase genes are involved in Mn(II) oxidation. Downregulation of SOS response, multidrug resistance and type Ⅳ secretion system related genes explained that Mn(II) and BioMnOx decreased HGT and mitigated resistance mutation by alleviating oxidative stress, which makes recipient cells more vulnerable to ARG acquisition and mutation. A manganese bio-oxidation based reactor was constructed and completely removed tetracycline with environmental concentration within 4-hour hydraulic retention time. Overall, this study suggests that Mn (II) bio-oxidation process could be exploited to control antibiotic contamination and mitigate resistance propagation during water treatment.

Feasibility of applying a noninvasive method for sleep monitoring based on mouse behaviors.

INTRODUCTION: Currently, electroencephalogram (EEG)/electromyogram (EMG) system is widely regarded as the "golden standard" for sleep monitoring. Imperfectly, its invasive monitoring may somehow interfere with the natural state of sleep. Up to now, noninvasive methods for sleep monitoring have developed, which could preserve the undisturbed and naïve sleep state of mice to the greatest extent, but the feasibility of their application under different conditions should be extensive validated. METHODS: Based on existing research, we verified the feasibility of a sleep monitoring system based on mouse behaviors under different conditions. The experimental mice were exposed to various stresses and placed into a combined device comprising noninvasive sleep monitoring equipment and an EEG/EMG system, and the sleep status was recorded under different physiological, pharmacological, and pathophysiological conditions. The consistency of the parameters obtained from the different systems was calculated using the Bland-Altman statistical method. RESULTS: The results demonstrated that the physiological sleep times determined by noninvasive sleep monitoring system were highly consistent with those obtained from the EEG/EMG system, and the coefficients were 94.4% and 95.1% in C57BL/6J and CD-1 mice, respectively. The noninvasive sleep monitoring system exhibited high sensitivity under the sleep-promoting effect of diazepam and caffeine-induced wakefulness, which was indicated by its ability to detect the effect of dosage on sleep times, and accurate determination of the sleep/wakeful status of mice under different pathophysiological conditions. After combining the data obtained from all the mice, the coefficient between the sleep times detected by behavior-based sleep monitoring system and those obtained from the EEG/EMG equipment was determined to .94. CONCLUSION: The results suggested that behavior-based sleep monitoring system could accurately evaluate the sleep/wakeful states of mice under different conditions.

Genomic alteration profile and PD-L1 expression among different breast cancer subtypes in Chinese population and their correlations.

BACKGROUD: There were limitations existing in programmed cell-death ligand 1 (PD-L1) as predictive biomarkers for breast cancer (BC), hence exploring the correlation between PD-L1 levels and other biomarkers in BC may become a very useful therapeutic clinical tool. METHODS: A total of 301 Chinese patients with different BC subtypes including 47 HR+/HER2+, 185 HR+/HER2-, 38 HR-/HER2+, and 31 triple-negative breast cancer (TNBC) were enrolled in our study. Next-generation sequencing based Yuansu450 gene panel was used for genomic alteration identification and PD-L1 expression was tested using immunohistochemistry. RESULTS: The most prevalent BC-related mutations were TP53 mutations, followed by mutations in PIK3CA, ERBB2, CDK12, and GATA3 in our Chinese cohort. We found that mutations DDR2 and MYCL were only mutated in HR-/HER2+ subtype, whereas H3-3A and NRAS mutations were only occurred in HR-/HER2- subtype. The percentage of patients with PD-L1-positive expression was higher in patients with HR-/HER2- mainly due to the percentage of PD-L1-high level. Mutational frequencies of TP53, MYC, FAT4, PBRM1, PREX2 were observed to have significant differences among patients with different BC subtypes based on PD-L1 levels. Moreover, a positive correlation was observed between TMB and PD-L1 level in HR+/HER2- subtype, and showed that the proportion of patients with high PD-L1 expression was higher than that of patients with low PD-L1 expression in the HR+/HER2- and HR+/HER2+ cohorts with high Ki67 expression. CONCLUSIONS: The genomic alterations based on PD-L1 and other biomarkers of different cohorts may provide more possibilities for the treatment of BC with different subtypes.

Single-cell profile of tumor and immune cells in primary breast cancer, sentinel lymph node, and metastatic lymph node.

PURPOSE: Little is known about the host-tumor interaction in the lymph-node basin at a single cell level. This study examines single cell sequences in breast cancer nodal metastases of a patient with triple-negative breast cancer. METHODS: The primary breast tumor, sentinel lymph node, an adjacent lymph node with metastatic involvement and a clinically normal-appearing lymph node were collected during surgery. Single-cell sequencing was performed on all four specimens. RESULTS: 14,016 cells were clustered into 6 cell subpopulations. Cancer cells demonstrated the molecular characteristics of TNBC basal B subtype and highly expressed genes in the MAPK signaling cascade. Tumor-associated macrophages regulated antigen processing and presentation and other immune-related pathways to promote tumor invasion. CD8 + and CD4 + T lymphocytes concentrated more in sentinel lymph node and mainly stratified into two transcriptional states. The immune-cell amount variation among primary tumor, sentinel and normal lymph nodes showed a similar tendency between the sc-RNA-seq profile of TNBC samples and a previous reported bulk RNA-seq profile of a breast cancer cohort, including all four breast cancer subtype samples. DISCUSSION: Single-cell sequencing analysis suggested that the sentinel lymph node was the initial meeting site of tumor infiltration and immune response, where partial T lymphocytes perform anti-tumor activity, while other T cells exhibit an exhausted state. We proposed a molecular explanation to the well-established clinical principle that the 5-year and 10-year survival outcomes were noninferior between SLND and ALND.

YTHDF1 amplification is correlated with worse outcome and lower immune cell infiltrations in breast cancer.

OBJECTIVE: N6-methyladenosine (m6A) is a common RNA modification on eukaryotic mRNA and some of the m6A regulatory proteins play a crucial role in breast cancer. However, the copy number variations for m6A regulatory proteins and their role in clinicopathological characteristics and survival in breast cancer remain unclear. METHODS: In this study, we screened the m6A related genes alterations in breast cancer by analyzing the Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas database, and further analyzed the clinical prognostic value of YTHDF1 amplification. RESULTS: The YTH domain family (YTHDF3 and YTHDF1) amplification exhibited higher alteration rates among 10 m6A regulatory genes. YTHDF1 and YTHDF3 amplification resulted in higher mRNA expression (P< 0.0001). Protein expression of YTHDF1 and YTHDF3 were higher in breast cancer (P< 0.0001). YTHDF1 amplification presented a high correlation with worse clinicopathological characteristics and overall survival in patients with breast cancer. Cox regression analysis showed that YTHDF1 amplification was an independent risk factor for 10-year overall survival in breast cancer (Hazard ratio: 1.663; 95% confidence interval: 1.298-2.131; P< 0.001). Gene set enrichment analysis revealed that the downstream target of YTHDF1 may be related to MYC signaling regulation and T cell differentiation. Moreover, YTHDF1 amplification and high expression resulted in lower immune cell infiltration. YTHDF1 knockdown retrained proliferation, migration and invasion in breast cancer cells in vitro. CONCLUSIONS: We found significant worse clinical characteristics and lower immune infiltrates in patients with YTHDF1 amplification. The findings indicate that YTHDF1 amplification may be a potential target for the treatment of breast cancer.

A postpartum enriched environment rescues impaired cognition and oxidative markers in aged mice with gestational inflammation.

INTRODUCTION: Previous studies have shown that gestational inflammation can accelerate age-associated cognitive decline (AACD) in maternal mice; enriched environments (EEs) have been reported to protect normally aging mice from AACD and improve mitochondrial function. However, it is unclear whether the nitrosative stress-related proteins tet methylcytosine dioxygenase 1 (TET1) and S-nitrosoglutathione reductase (GSNOR) are involved in the accelerated aging process of gestational inflammation and whether EEs can slow this process. METHODS: In this study, CD-1 female mice on the 15th day of pregnancy were injected with bacterial lipopolysaccharide (50 µg/kg; LPS group) or an equivalent amount of normal saline (CON group) from the abdominal cavity for 4 consecutive days. Twenty-one days after delivery, half of the LPS-treated mice were randomly selected for EE until the end of the behavioral experiment (LPS-E group). When the female rats were raised to 6 months and 18 months of age, the Morris water maze (MWM) was used to detect spatial learning and memory ability; RT-PCR and Western blots were used to measure the mRNA and protein levels of hippocampal TET1 and GSNOR. RESULTS: As for the control group, compared with 6-month-old mice, the spatial learning and memory ability of 18-month-old mice decreased, and the hippocampal TET1 and GSNOR mRNA and protein levels were decreased. Gestational inflammation exacerbated these age-related changes, but an EE alleviated the effects. Pearson's correlation analysis indicated that performance during the learning and memory periods in the MWM correlated with the levels of hippocampal TET1 and GSNOR. CONCLUSIONS: Our findings suggest that gestational inflammation accelerates age-related learning and memory impairments and that postpartum EE exposure could alleviate these changes. These effects may be related to hippocampal TET1 and GSNOR expression.

Characteristics of MYC Amplification and Their Association with Clinicopathological and Molecular Factors in Patients with Breast Cancer.

MYC amplification is detected in ∼15% of breast tumors and is associated with poor prognosis by mediating acquired resistance to anticancer therapies. This study aimed to determine the prevalence of MYC amplifications in Chinese women with breast cancer (BRCA) and investigate the correlation between MYC amplification and clinicopathological and molecular characteristics and its clinical implications. We analyzed MYC alterations in tissue specimens from 410 women diagnosed with BRCA in our hospital from June 1, 2017 to September 27, 2018. We compared our results with publicly available data from The Cancer Genome Atlas (TCGA) BRCA cohort (n = 1079). MYC amplification was identified in 12.4% (51/410) of our cohort, with mean copy number (CN) of 4.42 (range: 2.84-11.27). In TCGA cohort, MYC amplification was identified in 21.2% (229/1079) and was associated with age, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 (HER2) status, and molecular subtype, whereas in our cohort, MYC amplification was associated with smaller tumor size (T1-2, p = 0.023) and higher Ki-67 levels (≥20%; p = 0.031). Analysis of molecular profiles revealed that MYC-amplified breast tumors had significantly more concurrent CN variations compared with MYC nonamplified BRCA in both Guangdong Provincial People's Hospital (GDPH) and TCGA cohorts (p < 0.001). Pathway mapping analysis demonstrated that MYC-amplified tumors had more mutations involved in 15 different but interrelated pathways critical in DNA repair, cell cycle, and cell proliferation. Patients in TCGA cohort with MYC-amplified hormone receptor (HR)-positive/HER2-positive BRCA (p = 0.038) and MYC nonamplified triple-negative BRCA (p = 0.027) had significantly shorter overall survival. In conclusion, this study contributes to a better understanding that MYC-amplified breast tumors had distinct clinicopathological and molecular features compared with MYC nonamplified breast tumors. Further research with a larger sample size is necessary to further elucidate the clinical and survival implications of MYC amplifications.

Changes in telomere length and serum neurofilament light chain levels in female patients with chronic insomnia disorder.

STUDY OBJECTIVES: The aims of this study were to explore changes in the telomere length (relative telomere repeat copy/single-copy gene [T/S ratio]) and serum neurofilament light chain (sNfL) levels in female patients with chronic insomnia disorder (CID), examine their relationships with emotional abnormalities and cognitive impairment, and determine whether these 2 indicators were independently associated with sleep quality. METHODS: The CID group contained 80 patients diagnosed with CID, and 51 individuals constituted a healthy control group. Participants completed sleep, emotion, and cognition assessments. Telomere length was detected through quantitative real-time polymerase chain reaction. Enzyme-linked immunosorbent assay was used to determine sNfL concentrations. RESULTS: Relative to the healthy control group, the CID group had elevated Pittsburgh Sleep Quality Index, Hamilton Anxiety Scale-14, and Hamilton Depression Rating Scale-17 scores and reduced Montreal Cognitive Assessment scale scores, a decreased T/S ratio, and an increased sNfL concentration. Subgroup analysis according to various CID-associated sleep factors showed that poor sleep performance corresponded to a lower T/S ratio. Higher anxiety levels and more cognitive dysfunction correlated with shorter telomere lengths. The T/S ratio negatively correlated with age, whereas the sNfL concentration positively correlated with age in the CID group. The Pittsburgh Sleep Quality Index score negatively correlated with the T/S ratio but did not correlate with sNfL levels. Multiple linear regression analysis showed that the T/S ratio had a negative and independent effect on Pittsburgh Sleep Quality Index scores. CONCLUSIONS: The CID group had shorter telomeres and higher sNfL concentrations, and reduced telomere length independently affected sleep quality. CITATION: Ren C-Y, Liu P-P, Li J, et al. Changes in telomere length and serum neurofilament light chain levels in female patients with chronic insomnia disorder. J Clin Sleep Med. 2022;18(2):383-392.