Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

Exosomal circSIPA1L3-mediated intercellular communication contributes to glucose metabolic reprogramming and progression of triple negative breast cancer.

BACKGROUND: Breast cancer is the most common malignant tumor, and metastasis remains the major cause of poor prognosis. Glucose metabolic reprogramming is one of the prominent hallmarks in cancer, providing nutrients and energy to support dramatically elevated tumor growth and metastasis. Nevertheless, the potential mechanistic links between glycolysis and breast cancer progression have not been thoroughly elucidated. METHODS: RNA-seq analysis was used to identify glucose metabolism-related circRNAs. The expression of circSIPA1L3 in breast cancer tissues and serum was examined by qRT-PCR, and further assessed its diagnostic value. We also evaluated the prognostic potential of circSIPA1L3 by analyzing a cohort of 238 breast cancer patients. Gain- and loss-of-function experiments, transcriptomic analysis, and molecular biology experiments were conducted to explore the biological function and regulatory mechanism of circSIPA1L3. RESULTS: Using RNA-seq analysis, circSIPA1L3 was identified as the critical mediator responsible for metabolic adaption upon energy stress. Gain- and loss-of-function experiments revealed that circSIPA1L3 exerted a stimulative effect on breast cancer progression and glycolysis, which could also be transported by exosomes and facilitated malignant behaviors among breast cancer cells. Significantly, the elevated lactate secretion caused by circSIPA1L3-mediated glycolysis enhancement promoted the recruitment of tumor associated macrophage and their tumor-promoting roles. Mechanistically, EIF4A3 induced the cyclization and cytoplasmic export of circSIPA1L3, which inhibited ubiquitin-mediated IGF2BP3 degradation through enhancing the UPS7-IGF2BP3 interaction. Furthermore, circSIPA1L3 increased mRNA stability of the lactate export carrier SLC16A1 and the glucose intake enhancer RAB11A through either strengthening their interaction with IGF2BP3 or sponging miR-665, leading to enhanced glycolytic metabolism. Clinically, elevated circSIPA1L3 expression indicated unfavorable prognosis base on the cohort of 238 breast cancer patients. Moreover, circSIPA1L3 was highly expressed in the serum of breast cancer patients and exhibited high diagnostic value for breast cancer patients. CONCLUSIONS: Our study highlights the oncogenic role of circSIPA1L3 through mediating glucose metabolism, which might serve as a promising diagnostic and prognostic biomarker and potential therapeutic target for breast cancer.

CircTRIM1 encodes TRIM1-269aa to promote chemoresistance and metastasis of TNBC via enhancing CaM-dependent MARCKS translocation and PI3K/AKT/mTOR activation.

Peptides and proteins encoded by noncanonical open reading frames (ORFs) of circRNAs have recently been recognized to play important roles in disease progression, but the biological functions and mechanisms of these peptides and proteins are largely unknown. Here, we identified a potential coding circular RNA, circTRIM1, that was upregulated in doxorubicin-resistant TNBC cells by intersecting transcriptome and translatome RNA-seq data, and its expression was correlated with clinicopathological characteristics and poor prognosis in patients with TNBC. CircTRIM1 possesses a functional IRES element along with an 810 nt ORF that can be translated into a novel endogenously expressed protein termed TRIM1-269aa. Functionally, we demonstrated that TRIM1-269aa, which is involved in the biological functions of circTRIM1, promoted chemoresistance and metastasis in TNBC cells both in vitro and in vivo. In addition, we found that TRIM1-269aa can be packaged into exosomes and transmitted between TNBC cells. Mechanistically, TRIM1-269aa enhanced the interaction between MARCKS and calmodulin, thus promoting the calmodulin-dependent translocation of MARCKS, which further initiated the activation of the PI3K/AKT/mTOR pathway. Overall, circTRIM1, which encodes TRIM1-269aa, promoted TNBC chemoresistance and metastasis by enhancing MARCKS translocation and PI3K/AKT/mTOR activation. Our investigation has yielded novel insights into the roles of protein-coding circRNAs and supported circTRIM1/TRIM1-269aa as a novel promising prognostic and therapeutic target for patients with TNBC.

The radiological characteristics, tertiary lymphoid structures, and survival status associated with EGFR mutation in patients with subsolid nodules like stage I-II LUAD.

BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) recommended for the patients with subsolid nodule in early lung cancer stage is not routinely. The clinical value and impact in patients with EGFR mutation on survival outcomes is further needed to be elucidated to decide whether the application of EGFR-TKIs was appropriate in early lung adenocarcinoma (LUAD) stage appearing as subsolid nodules. MATERIALS AND METHODS: The inclusion of patients exhibiting clinical staging of IA-IIB subsolid nodules. Clinical information, computed tomography (CT) features before surgical resection and pathological characteristics including tertiary lymphoid structures of the tumors were recorded for further exploration of correlation with EGFR mutation and prognosis. RESULTS: Finally, 325 patients were enrolled into this study, with an average age of 56.8 ± 9.8 years. There are 173 patients (53.2%) harboring EGFR mutation. Logistic regression model analysis showed that female (OR = 1.944, p = 0.015), mix ground glass nodule (OR = 2.071, p = 0.003, bubble-like lucency (OR = 1.991, p = 0.003) were significant risk factors of EGFR mutations. Additionally, EGFR mutations were negatively correlated with TLS presence and density. Prognosis analysis showed that the presence of TLS was associated with better recurrence-free survival (RFS)(p = 0.03) while EGFR mutations were associated with worse RFS(p = 0.01). The RFS in patients with TLS was considerably excel those without TLS within EGFR wild type group(p = 0.018). Multivariate analyses confirmed that EGFR mutation was an independent prognostic predictor for RFS (HR = 3.205, p = 0.037). CONCLUSIONS: In early-phase LUADs, subsolid nodules with EGFR mutation had specific clinical and radiological signatures. EGFR mutation was associated with worse survival outcomes and negatively correlated with TLS, which might weaken the positive impact of TLS on prognosis. Highly attention should be paid to the use of EGFR-TKI for further treatment as agents in early LUAD patients who carrying EGFR mutation.

Long non-coding RNA MIDEAS-AS1 inhibits growth and metastasis of triple-negative breast cancer via transcriptionally activating NCALD.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with higher aggressiveness and poorer outcomes. Recently, long non-coding RNAs (lncRNAs) have become the crucial gene regulators in the progression of human cancers. However, the function and underlying mechanisms of lncRNAs in TNBC remains unclear. METHODS: Based on public databases and bioinformatics analyses, the low expression of lncRNA MIDEAS-AS1 in breast cancer tissues was detected and further validated in a cohort of TNBC tissues. The effects of MIDEAS-AS1 on proliferation, migration, invasion were determined by in vitro and in vivo experiments. RNA pull-down assay and RNA immunoprecipitation (RIP) assay were carried out to reveal the interaction between MIDEAS-AS1 and MATR3. Luciferase reporter assay, Chromatin immunoprecipitation (ChIP) and qRT-PCR were used to evaluate the regulatory effect of MIDEAS-AS1/MATR3 complex on NCALD. RESULTS: LncRNA MIDEAS-AS1 was significantly downregulated in TNBC, which was correlated with poor overall survival (OS) and progression-free survival (PFS) in TNBC patients. MIDEAS-AS1 overexpression remarkably inhibited tumor growth and metastasis in vitro and in vivo. Mechanistically, MIDEAS-AS1 mainly located in the nucleus and interacted with the nuclear protein MATR3. Meanwhile, NCALD was selected as the downstream target, which was transcriptionally regulated by MIDEAS-AS1/MATR3 complex and further inactivated NF-κB signaling pathway. Furthermore, rescue experiment showed that the suppression of cell malignant phenotype caused by MIDEAS-AS1 overexpression could be reversed by inhibition of NCALD. CONCLUSIONS: Collectively, our results demonstrate that MIDEAS-AS1 serves as a tumor-suppressor in TNBC through modulating MATR3/NCALD axis, and MIDEAS-AS1 may function as a prognostic biomarker for TNBC.

HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics.

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture (SILAC) and isobaric tandem mass tag (TMT) labeling enabled up to 28 single cells to be analyzed in a single liquid chromatography-mass spectrometry (LC-MS) analysis, in addition to carrier, reference, and negative control channels. A custom nested nanowell chip was used for nanoliter sample processing to minimize sample losses. Using a 145-min total LC-MS cycle time, ∼280 single cells were analyzed per day. This measurement throughput could be increased to ∼700 samples per day with a high-duty-cycle multicolumn LC system producing the same active gradient. The labeling efficiency and achievable proteome coverage were characterized for multiple analysis conditions.

circ-EIF6 encodes EIF6-224aa to promote TNBC progression via stabilizing MYH9 and activating the Wnt/beta-catenin pathway.

The protein-coding ability of circular RNAs (circRNAs) has recently been a hot topic, but the expression and roles of protein-coding circRNAs in triple-negative breast cancer (TNBC) remain uncertain. By intersecting circRNA sequencing data from clinical samples and cell lines, we identified a circRNA, termed circ-EIF6, which predicted a poorer prognosis and correlated with clinicopathological characteristics in a cohort of TNBC patients. Functionally, we showed that circ-EIF6 promoted the proliferation and metastasis of TNBC cells in vitro and in vivo. Mechanistically, we found that circ-EIF6 contains a 675-nucleotide (nt) open reading frame (ORF) and that the -150-bp sequence from ATG functioned as an internal ribosome entry site (IRES), which is required for translation initiation in 5' cap-independent coding RNAs. circ-EIF6 encodes a novel peptide, termed EIF6-224 amino acid (aa), which is responsible for the oncogenic effects of circ-EIF6. The endogenous expression of EIF6-224aa was further examined in TNBC cells and tissues by specific antibody. Moreover, EIF6-224aa directly interacted with MYH9, an oncogene in breast cancer, and decreased MYH9 degradation by inhibiting the ubiquitin-proteasome pathway and subsequently activating the Wnt/beta-catenin pathway. Our study provided novel insights into the roles of protein-coding circRNAs and supported circ-EIF6/EIF6-224aa as a novel promising prognostic and therapeutic target for tailored therapy in TNBC patients.

Data-Dependent Acquisition with Precursor Coisolation Improves Proteome Coverage and Measurement Throughput for Label-Free Single-Cell Proteomics.

We combined efficient sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify >3,000 proteins from single cells in rapid label-free analyses. WWA employs large isolation windows to intentionally co-isolate and co-fragment adjacent precursors along with the selected precursor. Optimized WWA increased the number of MS2-identified proteins by ≈40 % relative to standard data-dependent acquisition. For a 40-min LC gradient operated at ≈15 nL/min, we identified an average of 3,524 proteins per single-cell-sized aliquot of protein digest. Reducing the active gradient to 20 min resulted in a modest 10 % decrease in proteome coverage. Using this platform, we compared protein expression between single HeLa cells having an essential autophagy gene, atg9a, knocked out, with their isogenic WT parental line. Similar proteome coverage was observed, and 268 proteins were significantly up- or downregulated. Protein upregulation primarily related to innate immunity, vesicle trafficking and protein degradation.

In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50-200 µm.

Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories to cost-effectively preserve valuable specimens for later study. With the rapid growth of spatial proteomics, FFPE tissues can serve as a more accessible alternative to more commonly used frozen tissues. However, extracting proteins from FFPE tissues is challenging due to cross-links formed between proteins and formaldehyde. Here, we have adapted the nanoPOTS sample processing workflow, which was previously applied to single cells and fresh-frozen tissues, to profile protein expression from FFPE tissues. Following the optimization of extraction solvents, times, and temperatures, we identified an average of 1312 and 3184 high-confidence master proteins from 10 µm thick FFPE-preserved mouse liver tissue squares having lateral dimensions of 50 and 200 µm, respectively. The observed proteome coverage for FFPE tissues was on average 88% of that achieved for similar fresh-frozen tissues. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. This modified nanodroplet processing in one pot for trace samples (nanoPOTS) and fully automated processing in one pot for trace sample (autoPOTS) workflows provides the greatest coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues. Data are available via ProteomeXchange with identifier PXD029729.

Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform.

Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. We explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.

Metastatic heterogeneity of breast cancer: Molecular mechanism and potential therapeutic targets.

Breast cancer is one of the most common malignancies among women throughout the world and is the major cause of most cancer-related deaths. Several explanations account for the high rate of mortality of breast cancer, and metastasis to vital organs is identified as the principal cause. Over the past few years, intensive efforts have demonstrated that breast cancer exhibits metastatic heterogeneity with distinct metastatic precedence to various organs, giving rise to differences in prognoses and responses to therapy in breast cancer patients. Bone, lung, liver, and brain are generally accepted as the primary target sites of breast cancer metastasis. However, the underlying molecular mechanism of metastatic heterogeneity of breast cancer remains to be further elucidated. Recently, the advent of novel genomic and pathologic approaches as well as technological breakthroughs in imaging analysis and animal modelling have yielded an unprecedented change in our understanding of the heterogeneity of breast cancer metastasis and provided novel insight for establishing more effective therapeutics. This review summarizes recent molecular mechanisms and emerging concepts on the metastatic heterogeneity of breast cancer and discusses the potential of identifying specific molecules against tumor cells or tumor microenvironments to thwart the development of metastatic disease and improve the prognosis of breast cancer patients.

Salvianolate ameliorates oxidative stress and podocyte injury through modulation of NOX4 activity in db/db mice.

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and NOX4 is up-regulated in podocytes in response to high glucose. In the present study, the effects of Salvianolate on DN and its underlying mechanisms were investigated in diabetic db/db mice and human podocytes. We confirmed that the Salvianolate administration exhibited similar beneficial effects as the NOX1/NOX4 inhibitor GKT137831 treated diabetic mice, as reflected by attenuated albuminuria, reduced podocyte loss and mesangial matrix accumulation. We further observed that Salvianolate attenuated the increase of Nox4 protein, NOX4-based NADPH oxidase activity and restored podocyte loss in the diabetic kidney. In human podocytes, NOX4 was predominantly localized to mitochondria and Sal B treatment blocked HG-induced mitochondrial NOX4 derived superoxide generation and thereby ameliorating podocyte apoptosis, which can be abrogated by AMPK knockdown. Therefore, our results suggest that Sal B possesses the reno-protective capabilities in part through AMPK-mediated control of NOX4 expression. Taken together, our results identify that Salvianolate could prevent glucose-induced oxidative podocyte injury through modulation of NOX4 activity in DN and have a novel therapeutic potential for DN.

Breast cancer brain metastasis: insight into molecular mechanisms and therapeutic strategies.

Breast cancer is one of the most prevalent malignancies in women worldwide. Early-stage breast cancer is considered a curable disease; however, once distant metastasis occurs, the 5-year overall survival rate of patients becomes significantly reduced. There are four distinct metastatic patterns in breast cancer: bone, lung, liver and brain. Among these, breast cancer brain metastasis (BCBM) is the leading cause of death; it is highly associated with impaired quality of life and poor prognosis due to the limited permeability of the blood-brain barrier and consequent lack of effective treatments. Although the sequence of events in BCBM is universally accepted, the underlying mechanisms have not yet been fully elucidated. In this review, we outline progress surrounding the molecular mechanisms involved in BCBM as well as experimental methods and research models to better understand the process. We further discuss the challenges in the management of brain metastases, as well as providing an overview of current therapies and highlighting innovative research towards developing novel efficacious targeted therapies.

Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling.

Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 µm i.d. nanoLC columns. We used autoPOTS to analyze 1-500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.

Inhibition of p53/miR-34a/SIRT1 axis ameliorates podocyte injury in diabetic nephropathy.

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). MiR-34a, a p53-regulated miRNA, directly targets SIRT1 and contributed to DN progression. MiR-34a represses SIRT1 to activate p53 and establish a positive feedback loop. However, whether p53/miR-34a/SIRT1 signaling is activated in podocytes and contributes to DN pathogenesis remains elusive. In this study, we observed that serum miR-34a level was positively correlated with podocyte injury in DN patients. The expression of acetylated p53 and miR-34a was upregulated, SIRT1was downregulated in glomeruli from patients with DN and STZ induced diabetic mice, as well as in human podocytes treated with advanced glycation end (AGE). MiR-34a antagonism in vitro and vivo in STZ induced diabetic mice developed alleviated glomerulus injury as reflected by attenuated albuminuria, reduced podocyte loss and restored autophagic flux. In human podocyte, inhibition of AGE formation by pyridoxamine prevented miR-34a dependent repression of SIRT1, p53 acetylation and activate podocyte autophagy in a dose-dependent manner. MiR-34a overexpression increases acetylation of p53 by translational repression of SIRT1. SIRT1 overexpression also impacts AGE induced apoptosis through deacetylating p53, whereas silencing of SIRT1 by EX527 attenuated the cytoprotective functions of miR-34a knockdown. Moreover, blockade of p53 acetylation significantly rescued miR-34a-induced apoptosis through SIRT1 restoration. Collectively, we demonstrate that by activation of p53, AGE induced the transcription of miR-34a, miR-34a in turn repressed SIRT1 to activate p53, resulting in a positive-feedback loop and contributing to podocyte injury. Targeting modulation of p53/miR-34a/SIRT1 feedback by miR-34a knockdown or overexpression of SIRT1 could rescue podocyte injury during DN.

Depletion of miR-21 in dendritic cells aggravates renal ischemia-reperfusion injury.

Dendritic cells (DCs) play an important role in the pathophysiology of renal ischemia-reperfusion injury (IRI). The mechanisms underlying DCs phenotypic modulation and their function are not fully understood. In this study, we examined the effect of miR-21 on the phenotypic modulation of DCs in vitro and in vivo, and further investigated the impact of miR-21-overexpression DC or miR-21-deficient DC on renal IRI. We found that treatment with hypoxia/reoxygenation (H/R) suppressed miR-21 expression in bone marrow-derived dendritic cells (BMDCs), and significantly increased the percentage of mature DCs (CD11c+ /MHC-II+ /CD80+ ). Using a selection of microRNA mimics, we successfully induced the upregulation of miR-21 in BMDCs, which induced immature DC phenotype and an anti-inflammatory DC response. However, deletion of miR-21 in BMDCs promoted maturation of DCs under H/R. Adoptive transfer of miR-21-overexpression BMDCs could alleviate renal IR-induced pro-inflammatory cytokines production and acute kidney injury (AKI). Mice with miR-21 deficiency in DCs subjected to renal IR showed more severe renal dysfunction and inflammatory response compared with wild-type mice. In addition, chemokine C receptor 7 (CCR7), a surface marker of mature DC, was a target gene of miR-21, and silencing of CCR7 in BMDCs led to reduced mature DCs under H/R. In conclusion, our findings highlight miR-21 as a key regulator of DCs subset phenotype and a potential therapeutic target in renal IRI.

Application of preoperative computed tomographic lymphography for precise sentinel lymph node biopsy in breast cancer patients.

BACKGROUND: In light of the extensive application of sentinel lymph node biopsy (SLNB) in clinically node-negative breast cancer patients and the recently investigated failure of SLNB after lumpectomy, it has become important to explore methods for preoperative mapping of sentinel lymph nodes (SLNs) and their lymphatics to direct precise SLNB and improve the identification rate of SLNs. METHODS: Twenty-seven patients with suspected breast cancer based on the results of the clinical examination and imaging were enrolled in the study. Computed tomographic lymphography (CTLG) followed by CT three-dimensional reconstruction was performed to determine the localization of SLNs and lymphatics on the body surface preoperatively. Intraoperatively combined staining with methylene blue and indocyanine green was used to evaluate the accuracy and feasibility of CTLG. RESULTS: SLNs and lymphatics from the breast were identified using CTLG in all patients, and preoperative SLNs and lymphatics localization on the body surface showed a significant role in the selection of operative incision and injection points. The accuracy rate of SLN and lymphatic detection by CTLG was 92.6% compared with intraoperatively combined staining. Moreover, preoperative CTLG performed well in SLN number detection, and the accuracy rate was 95.2%. CONCLUSION: We evaluate the procedure and application of preoperative CTLG in the superficial localization of SLNs and lymphatics, which may lead to a decreased incidence of cutting off the lymphatics of SLNs and consequently more rapid and accurate SLN detection. This method promotes personalized SLN mapping, providing detailed information about the number and anatomical location of SLNs and lymphatics for adequate surgical planning for breast cancer patients.

Terminal residue and dietary intake risk assessment of prothioconazole-desthio and fluoxastrobin in wheat field ecosystem.

BACKGROUND: Wheat is one of the most important cereal crops worldwide, and use of fungicides is an essential part of wheat production. Both prothioconazole and fluoxastrobin give excellent control of important seed and soilborne pathogens. The combination of these two fungicides shows a complementary mode of action and has a wide usage around the world. But the residue levels of these fungicides in the wheat matrix are still unknown. RESULTS: In the current study, a simple, low-cost and highly sensitive method using modified QuECHERS procedure combined with high-performance liquid chromatography-tandem mass spectrometry was developed to simultaneously quantify E- and Z-fluoxastrobin and the main metabolite prothioconazole-desthio of prothioconazole in the wheat matrix. The recoveries of prothioconazole-desthio, E-fluoxastrobin and Z-fluoxastrobin ranged from 84% to 101%, with relative standard deviation of less than 13.2%. The terminal residues of prothioconazole-desthio and E- and Z-fluoxastrobin were studied in wheat grain and straw under field conditions. The results showed that the terminal residue of the target compounds ranged from <0.01 to 0.029 mg kg-1 and <0.05 to 7.6 mg kg-1 in wheat grain and straw (expressed as dry weight), respectively. The risk quotients of prothioconazole-desthio and fluoxastrobin were 0.2% and 3.2%. CONCLUSIONS: The residue levels of the target analytes in wheat grain were lower than the maximum residue limits recommended by the Chinese Ministry of Agriculture. And the calculated risk quotient values were far below 100%, indicating a low dietary intake health risk to consumers. © 2021 Society of Chemical Industry.

LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis.

BACKGROUND: Long noncoding RNAs (lncRNAs) play crucial roles in tumor progression and are aberrantly expressed in various cancers. However, the functional roles of lncRNAs in breast cancer remain largely unknown. METHODS: Based on public databases and integrating bioinformatics analyses, the overexpression of lncRNA BCRT1 in breast cancer tissues was detected and further validated in a cohort of breast cancer tissues. The effects of lncRNA BCRT1 on proliferation, migration, invasion and macrophage polarization were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA BCRT1, miR-1303, and PTBP3. Chromatin immunoprecipitation (ChIP) and RT-PCR were used to evaluate the regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on lncRNA BCRT1. RESULTS: LncRNA BCRT1 was significantly upregulated in breast cancer tissues, which was correlated with poor prognosis in breast cancer patients. LncRNA BCRT1 knockdown remarkably suppressed tumor growth and metastasis in vitro and in vivo. Mechanistically, lncRNA BCRT1 could competitively bind with miR-1303 to prevent the degradation of its target gene PTBP3, which acts as a tumor-promoter in breast cancer. LncRNA BCRT1 overexpression could promote M2 polarization of macrophages, mediated by exosomes, which further accelerated breast cancer progression. Furthermore, lncRNA BCRT1 was upregulated in response to hypoxia, which was attributed to the binding of HIF-1α to HREs in the lncRNA BCRT1 promoter. CONCLUSIONS: Collectively, these results reveal a novel HIF-1α/lncRNA BCRT1/miR-1303/PTBP3 pathway for breast cancer progression and suggest that lncRNA BCRT1 might be a potential biomarker and therapeutic target for breast cancer.

Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry.

Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 µm i.d. in-house-packed nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ∼20 nL/min flow rate provided by the LC separation. Ultrasensitive LC-MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 µm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.