SMMP: A Deep-Coverage Marine Metaproteome Method for Microbial Community Analysis throughout the Water Column Using 1 L of Seawater.

Marine microbes drive pivotal transformations in planetary-scale elemental cycles and have crucial impacts on global biogeochemical processes. Metaproteomics is a powerful tool for assessing the metabolic diversity and function of marine microbes. However, hundreds of liters of seawater are required for normal metaproteomic analysis due to the sparsity of microbial populations in seawater, which poses a substantial challenge to the widespread application of marine metaproteomics, particularly for deep seawater. Herein, a sensitive marine metaproteomics workflow, named sensitive marine metaproteome analysis (SMMP), was developed by integrating polycarbonate filter-assisted microbial enrichment, solid-phase alkylation-based anti-interference sample preparation, and narrow-bore nanoLC column for trace peptide separation and characterization. The method provided more than 8500 proteins from 1 L of bathypelagic seawater samples, which covered diverse microorganisms and crucial functions, e.g., the detection of key enzymes associated with the Wood-Ljungdahl pathway. Then, we applied SMMP to investigate vertical variations in the metabolic expression patterns of marine microorganisms from the euphotic zone to the bathypelagic zone. Methane oxidation and carbon monoxide (CO) oxidation were active processes, especially in the bathypelagic zone, which provided a remarkable energy supply for the growth and proliferation of heterotrophic microorganisms. In addition, marker protein profiles detected related to ammonia transport, ammonia oxidation, and carbon fixation highlighted that Thaumarchaeota played a critical role in primary production based on the coupled carbon-nitrogen process, contributing to the storage of carbon and nitrogen in the bathypelagic regions. SMMP has low microbial input requirements and yields in-depth metaproteome analysis, making it a prospective approach for comprehensive marine metaproteomic investigations.

The protease corin regulates electrolyte homeostasis in eccrine sweat glands.

Sweating is a basic skin function in body temperature control. In sweat glands, salt excretion and reabsorption are regulated to avoid electrolyte imbalance. To date, the mechanism underlying such regulation is not fully understood. Corin is a transmembrane protease that activates atrial natriuretic peptide (ANP), a cardiac hormone essential for normal blood volume and pressure. Here, we report an unexpected role of corin in sweat glands to promote sweat and salt excretion in regulating electrolyte homeostasis. In human and mouse eccrine sweat glands, corin and ANP are expressed in the luminal epithelial cells. In corin-deficient mice on normal- and high-salt diets, sweat and salt excretion is reduced. This phenotype is associated with enhanced epithelial sodium channel (ENaC) activity that mediates Na+ and water reabsorption. Treatment of amiloride, an ENaC inhibitor, normalizes sweat and salt excretion in corin-deficient mice. Moreover, treatment of aldosterone decreases sweat and salt excretion in wild-type (WT), but not corin-deficient, mice. These results reveal an important regulatory function of corin in eccrine sweat glands to promote sweat and salt excretion.

Construction and Validation of a Prognostic Risk Prediction Model for Lactate Metabolism-Related lncRNA in Endometrial Cancer.

Endometrial cancer (EC) is a common group of malignant epithelial tumors that mainly occur in the female endometrium. Lactate is a key regulator of signal pathways in normal and malignant tissues. However, there is still no research on lactate metabolism-related lncRNA in EC. Here, we intended to establish a prognostic risk model for EC based on lactate metabolism-related lncRNA to forecast the prognosis of EC patients. First, we found that 38 lactate metabolism-associated lncRNAs were significantly overall survival through univariate Cox regression analysis. Using minimum absolute contraction and selection operator (LASSO) regression analysis and multivariate Cox regression analysis, six lactate metabolism-related lncRNAs were established as independent predictor in EC patients and were used to establish a prognostic risk signature. We next used multifactorial COX regression analysis and receiver operating characteristic (ROC) curve analysis to confirm that risk score was an independent prognostic factor of overall patient survival. The survival time of patients with EC in different high-risk populations was obviously related to clinicopathological factors. In addition, lactate metabolism-related lncRNA in high-risk population participated in multiple aspects of EC malignant progress through Gene Set Enrichment Analysis, Genomes pathway and Kyoto Encyclopedia of Genes and Gene Ontology. And risk scores were strongly associated with tumor mutation burden, immunotherapy response and microsatellite instability. Finally, we chose a lncRNA SRP14-AS1 to validate the model we have constructed. Interestingly, we observed that the expression degree of SRP14-AS1 was lower in tumor tissues of EC patients than in normal tissues, which was consistent with our findings in the TCGA database. In conclusion, our study constructed a prognostic risk model through lactate metabolism-related lncRNA and validated the model, confirming that the model can be used to predict the prognosis of EC patients and providing a molecular analysis of potential prognostic lncRNA for EC.

Spatial and temporal distribution characteristics and risk assessment of heavy metals in groundwater of Pingshuo mining area.

Groundwater pollution in the Pingshuo mining area is strongly associated with mining activities, with heavy metals (HMs) representing predominant pollutants. To obtain accurate information about the pollution status and health risks of groundwater, 189 groups of samples were collected from four types of groundwater, during three periods of the year, and analyzed for HMs. The results showed that the concentration of HMs in groundwater was higher near the open pit, waste slag pile, riverfront area, and human settlements. Except for Ordovician groundwater, excessive HMs were found in all investigated groundwater of the mining area, as compared with the standard thresholds. Fe exceeded the threshold in 13-75% of the groundwater samples. Three sources of HMs were identified and quantified by Pearson's correlation analysis and the PMF model, including coal mining activities (68.22%), industrial, agricultural, and residential chemicals residue and leakage (16.91%), and natural sources (14.87%). The Nemerow pollution index revealed that 7.58% and 100% of Quaternary groundwater and mine water samples were polluted. The health risk index for HMs in groundwater showed that the non-carcinogenic health risk ranged from 0.18 to 0.42 for adults, indicating an acceptable level. Additionally, high carcinogenic risks were identified in Quaternary groundwater (95.45%), coal series groundwater (91.67%), and Ordovician groundwater (26.67%). Both carcinogenic and non-carcinogenic risks were greater for children than adults, highlighting their increased vulnerability to HMs in groundwater. This study provides a scientific foundation for managing groundwater quality and ensuring drinking water safety in mining areas.

Krüppel-like factor 17 upregulates uterine corin expression and promotes spiral artery remodeling in pregnancy.

Spiral artery remodeling is an important physiological process in the pregnant uterus which increases blood flow to the fetus. Impaired spiral artery remodeling contributes to preeclampsia, a major disease in pregnancy. Corin, a transmembrane serine protease, is up-regulated in the pregnant uterus to promote spiral artery remodeling. To date, the mechanism underlying uterine corin up-regulation remains unknown. Here we show that Krüppel-like factor (KLF) 17 is a key transcription factor for uterine corin expression in pregnancy. In cultured human uterine endometrial cells, KLF17 binds to the CORIN promoter and enhances the promoter activity. Disruption of the KLF17 gene in the endometrial cells abolishes CORIN expression. In mice, Klf17 is up-regulated in the pregnant uterus. Klf17 deficiency prevents uterine Corin expression in pregnancy. Moreover, Klf17-deficient mice have poorly remodeled uterine spiral arteries and develop gestational hypertension and proteinuria. Together, our results reveal an important function of KLF17 in regulating Corin expression and uterine physiology in pregnancy.

Anti-diabetic drug canagliflozin hinders skeletal muscle regeneration in mice.

Canagliflozin is an antidiabetic medicine that inhibits sodium-glucose cotransporter 2 (SGLT2) in proximal tubules. Recently, it was reported to have several noncanonical effects other than SGLT2 inhibiting. However, the effects of canagliflozin on skeletal muscle regeneration remain largely unexplored. Thus, in vivo muscle contractile properties recovery in mice ischemic lower limbs following gliflozins treatment was evaluated. The C2C12 myoblast differentiation after gliflozins treatment was also assessed in vitro. As a result, both in vivo and in vitro data indicate that canagliflozin impairs intrinsic myogenic regeneration, thus hindering ischemic limb muscle contractile properties, fatigue resistance recovery, and tissue regeneration. Mitochondrial structure and activity are both disrupted by canagliflozin in myoblasts. Single-cell RNA sequencing of ischemic tibialis anterior reveals a decrease in leucyl-tRNA synthetase 2 (LARS2) in muscle stem cells attributable to canagliflozin. Further investigation explicates the noncanonical function of LARS2, which plays pivotal roles in regulating myoblast differentiation and muscle regeneration by affecting mitochondrial structure and activity. Enhanced expression of LARS2 restores the differentiation of canagliflozin-treated myoblasts, and accelerates ischemic skeletal muscle regeneration in canagliflozin-treated mice. Our data suggest that canagliflozin directly impairs ischemic skeletal muscle recovery in mice by downregulating LARS2 expression in muscle stem cells, and that LARS2 may be a promising therapeutic target for injured skeletal muscle regeneration.

Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease.

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.

The Stock Market Model with Delayed Information Impact from a Socioeconomic View.

Finding the critical factor and possible "Newton's laws" in financial markets has been an important issue. However, with the development of information and communication technologies, financial models are becoming more realistic but complex, contradicting the objective law "Greatest truths are the simplest." Therefore, this paper presents an evolutionary model independent of micro features and attempts to discover the most critical factor. In the model, information is the only critical factor, and stock price is the emergence of collective behavior. The statistical properties of the model are significantly similar to the real market. It also explains the correlations of stocks within an industry, which provides a new idea for studying critical factors and core structures in the financial markets.

Lipofectamine 2000/siRNA complexes cause endoplasmic reticulum unfolded protein response in human endothelial cells.

Lipofectamine 2000 (Lipo2000) delivery system is commonly used for short interfering RNA (siRNA) transfection, whereas the cellular responses have attracted little attention. The purpose of this study is to evaluate the effect of siRNA transfection using Lipo2000 on cellular functions and the possible underlying mechanism. Primary human umbilical vein endothelial cells (HUVECs) and adult human coronary artery endothelial cell line (HCAECs) were treated with different concentrations of a Lipo2000/negative control siRNA (NC siRNA) complex or Lipo2000 for specific durations. The cell proliferation, apoptosis rate, and protein expression of claudin5 (CLDN5) and ETS-related gene (ERG) were analyzed as indicators of cellular function. The effects of the Lipo2000/NC siRNA complex on cellular autophagy and endoplasmic reticulum (ER) unfolded protein response (UPR) were investigated by western blot and real-time polymerase chain reaction analyses; autophagy was also evaluated by transmission electron microscopy. The Lipo2000/NC siRNA complex inhibited proliferation, downregulated various proteins, and increased the apoptosis in both HUVECs and HCAECs. Both autophagy and UPR were observed in HUVECs treated with the Lipo2000/NC siRNA complex, ER stress-induced autophagy acted as a cellular protective factor against apoptosis, as inhibition of autophagy by chemical inhibitors increased the cell apoptosis rate. Chemical chaperones failed to prevent the Lipo2000/siRNA complex-induced UPR. However, knockdown of protein kinase RNA-like ER kinase and inositol-requiring protein 1, instead of activating transcription factor-6, partially ameliorated the UPR and reversed the protein level of CLDN5 and ERG downregulated by Lipo2000/NC siRNA complex. This study provides the first evidence that the Lipo2000-mediated transport of siRNA leads to an increase in UPR and ER stress-related apoptosis in endothelial cells.

Synthesis of Indoles through Domino Reactions of 2-Fluorotoluenes and Nitriles.

Indoles are essential heterocycles in medicinal chemistry, and therefore, novel and efficient approaches to their synthesis are in high demand. Among indoles, 2-aryl indoles have been described as privileged scaffolds. Advanced herein is a straightforward, practical, and transition-metal-free assembly of 2-aryl indoles. Simply combining readily available 2-fluorotoluenes, nitriles, LiN(SiMe3 )2 , and CsF enables the generation of a diverse array of indoles (38 examples, 48-92 % yield). A range of substituents can be introduced into each position of the indole backbone (C4 to C7, and aryl groups at C2), providing handles for further elaboration.

High molecular weight fibroblast growth factor-2 as a promising prognostic biomarker to predict the occurrence of heart failure in atrial fibrillation patients.

Heart failure (HF) has a significant effect on the prognosis of the patients with atrial fibrillation (AF), and also it is an important risk factor for overall mortality. High molecular weight fibroblast growth factor-2 (Hi-FGF-2) is emerging as a prognostic marker with HF and AF. The aim of this study was to prove that Hi-FGF-2 would predict occurrence of HF in the patients with AF. Subjects diagnosed with paroxysmal AF (Group paAF), persistent AF (Group peAF) and sinus rhythm (Group SR) were enrolled in the study. Serum Hi-FGF-2 concentration was measured by ELISA at baseline. Multivariable logistic models and receiver operating characteristic (ROC) curve analysis were established to predict the prognosis of AF subjects. 260 patients were enrolled in the study: 104 (40.0%) admitted for sinus rhythm (Group SR) and 156 (60.0%) with AF (Group paAF and Group peAF). The Hi-FGF-2 levels were much lower in the Group SR (58.2 ± 27.1 ng/L) than in the Group AF. Furthermore, the Group peAF (84.3 ± 34.1 ng/L) had higher Hi-FGF-2 levels than the Group paAF (72.9 ± 35.8 ng/L). Serum Hi-FGF-2 levels were classified into trisection in the multivariable logistic model (T1 < 57.3 ng/L, 57.3 < T2 < 86.5 ng/L, and T3 > 86.5 ng/L). Hi-FGF-2 showed good predictive ability for new-onset HF in the patients with AF. The occurrence of HF was associated significantly with increased tertile of serum Hi-FGF-2 levels (T2: OR 5.922, 95% CI 1.109-31.626, P = 0.037 and T3: OR 8.262, 95% CI 1.735-39.343, P = 0.008). ROC curve analysis showed that the area under curves for Hi-FGF-2 were 0.720 (P < 0.0001). Hi-FGF-2 has a significant meaning in AF subjects. Further to this, higher circulating Hi-FGF-2 was highly related to persistent AF, and Hi-FGF-2 may be an independent risk factor of occurrence HF in AF subjects.

Wicking Enhancement in Three-Dimensional Hierarchical Nanostructures.

Wicking, the absorption of liquid into narrow spaces without the assistance of external forces, has drawn much attention due to its potential applications in many engineering fields. Increasing surface roughness using micro/nanostructures can improve capillary action to enhance wicking. However, reducing the structure length scale can also result in significant viscous forces to impede wicking. In this work, we demonstrate enhanced wicking dynamics by using nanostructures with three-dimensional (3D) hierarchical features to increase the surface area while mitigating the obstruction of liquid flow. The proposed structures were engineered using a combination of interference lithography and hydrothermal synthesis of ZnO nanowires, where structures at two length scales were independently designed to control wicking behavior. The fabricated hierarchical 3D structures were tested for water and ethanol wicking properties, demonstrating improved wicking dynamics with intermediate nanowire lengths. The experimental data agree with the derived fluid model based on the balance of capillary and vicious forces. The hierarchical wicking structures can be potentially used in applications in water harvesting surfaces, microfluidics, and integrated heat exchangers.

Circulating Hepatocellular Carcinoma Cells are Characterized by CXCR4 and MMP26.

BACKGROUND/AIMS: Primary hepatocellular carcinoma (HCC) is highly invasive, and often results in an early distal metastasis resulting in poor prognosis and therapeutic outcome. Cancer cells disseminating from the tumor and entering circulation are termed circulating tumor cells (CTCs). Although substantial progress has been made to identify those CTCs in HCC, no good marker (cocktail) has so far been identified. METHODS: Since only tumorigenic CTCs form metastatic tumor in distal organs, we thus compared the HCC cells that form tumor spheres in culture to those that do not. We transduced HCC cells with a RFP reporter under MMP26 promoter and purified MMP26+CXCR4+ HCC cells. We examined tumor sphere formation in culture, presence of tumor cells in the circulation as well as capability of developing metastatic tumor after transplantation of MMP26+CXCR4+ HCC cells into nude mice, compared to other populations in HCC. RESULTS: Sphere-forming HCC cells expressed high levels of MMP26 and CXCR4. MMP26+CXCR4+ HCC cells formed significantly more tumor spheres in culture, compared to MMP26-CXCR4-, MMP26-CXCR4+ or MMP26+CXCR4- HCC cells. Moreover, tumor cells were more frequently detected in the circulation when MMP26+CXCR4+ HCC cells were subcutaneously transplanted. Further, subcutaneous transplantation of MMP26+CXCR4+ HCC cells, but not transplantation of MMP26-CXCR4-, MMP26-CXCR4+ or MMP26+CXCR4-HCC cells significantly developed distal metastatic tumors. CONCLUSION: MMP26+CXCR4+ cells may be CTCs in HCC. Selective elimination of MMP26+CXCR4+ cells may substantially reduce HCC metastasis after primary tumor resection.

Trajectories of Body Mass Index and Risk for Diabetes Complications and All-Cause Mortality in Finnish Type 2 Diabetes Patients.

Objective: We aimed to assess how longitudinal body mass index (BMI) trajectories are associated with diabetes complications and all-cause mortality in Finnish patients with type 2 diabetes (T2D). Methods: In this cohort study, electronic health records from public primary and specialized healthcare services in all 13 municipalities of North Karelia, Finland, were utilized. This study included a total of 889 adults with newly diagnosed T2D in 2011 or 2012 (mean age at baseline 62.0 years). Individual BMI trajectories from the T2D diagnosis until 2014 were estimated and grouped by growth mixture modeling (GMM). Hazard ratios (HRs) with 95% confidence intervals (CIs) for microvascular complications, macrovascular complications, any diabetes complications, and all-cause mortality from 2015 to 2022 across BMI trajectory groups were estimated using Cox regression models. Results: Three distinct BMI trajectory groups were identified using GMM and labeled as follows: "stable" (n = 774, 87.1%), "decreasing" (n = 87, 9.8%), and "increasing" (n = 28, 3.1%). During a median follow-up of 8 years, there were 119 (13.3%) patients with microvascular complications, 187 (21.0%) with macrovascular complications, 258 (29.0%) with any diabetes complications, and 180 (20.2%) deaths. Compared with the "stable" BMI, the "increasing" BMI was associated with an increased risk of microvascular complications (HR = 2.88, 95% CI: 1.32 to 6.28), macrovascular complications (HR = 2.52, 95% CI: 1.17 to 5.43), and any diabetes complications (HR = 2.21, 95% CI: 1.16 to 4.20). The "decreasing" BMI was associated with an increased risk of all-cause mortality (HR = 1.90, 95% CI: 1.14 to 3.15), compared to the "stable" BMI. Conclusion: Our findings underscore the significance of continuous BMI monitoring and weight management in patients with T2D. Tailored treatments are crucial for efficiently preventing weight gain and reducing the risk of diabetes complications.

Clinicopathological Features of CCDC6-RET and NCOA4-RET Fusions in Thyroid Cancer: A Single-center Retrospective Cohort Study in a Chinese Population.

BACKGROUND: The rearranged during transfection (RET) proto-oncogene fusion is common in papillary thyroid cancer (PTC), varying across ethnic groups. However, comprehensive comparisons of RET fusion types are limited. This study aims to identify predominant RET fusions and analyze their clinicopathological characteristics in a cohort of Chinese thyroid cancer cases. METHODS: This single-center retrospective cohort study analyzed thyroid cancer data, utilizing next-generation sequencing on formalin-fixed, paraffin-embedded tissue samples. Detailed clinicopathological data of thyroid cancer cases with RET fusions were collected. RESULTS: Among 2300 thyroid cancer cases, RET fusions were exclusively found in PTC or differentiated high-grade thyroid carcinoma (DHGTC) cases (2234 cases), absent in other types (66 cases). Of the 2234 PTC or DHGTC cases, 113 (5.06%) exhibited RET fusions, including 100 primary cases. CCDC6-RET fusions predominated (78.0%, 78/100), with NCOA4-RET fusions representing 22.0% (22/100). NCOA4-RET fusions were more prevalent in patients aged 45 years and older (54.5% vs. 28.2%, P = 0.021) and DHGTC cases (P < 0.05), and associated with higher rates of lymph node metastases (90.9% vs. 67.9%, P = 0.032). CCDC6-RET fusion exhibited a higher prevalence of Hashimoto's thyroiditis (67.9% vs. 22.7%, P < 0.001) and elevated thyroglobulin antibody levels (14.11 [1.86-174.32] IU/mL vs. 2.01 [1.14-15.41] IU/mL, P = 0.018). Moreover, CCDC6-RET fusion predominantly occurred in classic PTC (56.4%, 44/78) and infiltrative follicular PTC (17.9%, 14/78), whereas NCOA4-RET fusion was more frequent in classic PTC (36.4%, 8/22), solid PTC (27.3%, 6/22), and DHGTC (27.3%, 6/22). RET fusions with compound mutations were associated with older age (≥45 years) and bilateral thyroid involvement. Follow-up data showed a higher recurrence rate in the RET fusion group compared to the BRAF V600E mutation group (5.0% vs. 0.0%, P = 0.018). Although the NCOA4-RET group showed a numerically higher recurrence rate compared to CCDC6-RET (9.1% vs. 3.8%), this difference was not statistically significant (P = 0.559). CONCLUSIONS: RET fusions are specific to PTC or DHGTC cases among Chinese thyroid cancer cases. CCDC6-RET and NCOA4-RET fusions exhibited distinct clinicopathological features, with NCOA4-RET being more aggressive.

Multi-Model Strategies for Prevention of Infection Caused by Certain Multi-Drug Resistant Organisms in A Rehabilitation Unit: A Semi-Experimental Study.

Objective: To assess the effectiveness of multi-model strategies on healthcare-associated infections (HAIs) caused by multi-drug resistant organisms (MDROs) in rehabilitation units. Methods: A semi-experimental study was conducted in a rehabilitation unit with 181 beds from January 2021 to December 2022 in a teaching hospital with 4300 beds in China. In 2021, many basic prevention and control measures were conducted routinely. Based on the basic measures, strengthening multi-model strategies for the prevention and control of MDROs was pursued year-round since 1 January 2022. Results: A total of 6206 patients were enrolled during the study period. The incidence density of HAIs caused by MDROs decreased from 1.22 (95% CI, 0.96~1.54) cases/1000 patient-days in the pre-intervention period to 0.70 (95% CI, 0.50~0.95) cases/1000 patient-days (p = 0.004). Similarly, the incidence of HAIs in the intervention period was 50.85% lower than that in the pre-intervention period (2.02 (95% CI, 1.50~2.72) vs. 4.11 (95% CI, 3.45-4.85) cases/100 patients, p < 0.001). The rate of MDROs isolated from the environment decreased by 30.00%, although the difference was not statistically significant (p = 0.259). Conclusion: Multi-model strategies can reduce the incidence of HAIs and HAIs caused by certain MDROs in the rehabilitation unit.

Diagnostic performance of next-generation sequencing and genetic profiling in thyroid nodules from a single center in China.

Objective: The data regarding the mutation landscape in Chinese patients with thyroid cancer are limited. The diagnostic performance of thyroid nodules by fine-needle aspiration (FNA) cytology needs optimization, especially in indeterminate nodules. Methods: A total of 1039 FNA and surgical resection samples tested using the targeted multigene next-generation sequencing (NGS) panel were retrospectively collected. The features of gene alterations in different thyroid tumors were analyzed, and the diagnostic efficacy was evaluated. Results: Among 1039 samples, there were 822 FNA and 217 surgical FFPE samples. Among 207 malignant thyroid resections, a total of 181 out of 193 papillary thyroid carcinomas (PTCs) were NGS-positive (93.8%), with a high prevalence of BRAF mutations (81.9%, 158/193) and a low prevalence of RAS (1.0%, 2/193) and TERT promoter mutations (3.6%, 7/193). Gene fusions, involving the RET and NTRK3 genes, were present in 20 PTCs (10.4%) and mutually exclusive with other driver mutations. Two of three follicular thyroid carcinomas harbored multiple mutations. RET gene point mutations were common in medullary thyroid carcinoma (8/11, 72.7%). The combination of cytology and DNA-RNA-based NGS analysis demonstrated superior diagnostic value (98.0%) in FNA samples. For indeterminate thyroid nodules, the diagnostic sensitivity and specificity of NGS testing were 79.2 (38/48) and 80.0% (8/10), respectively. Two mutation-positive benign cases harbored NRAS and TSHR mutations, respectively. Conclusions: Our study revealed the distinct molecular profile of thyroid tumors in the Chinese population. The combination of NGS testing and FNA cytology could facilitate the accurate diagnosis of thyroid nodules, especially for indeterminate nodules.

A photo-oxidation driven proximity labeling strategy enables profiling of mitochondrial proteome dynamics in living cells.

Mapping the proteomic landscape of mitochondria with spatiotemporal precision plays a pivotal role in elucidating the delicate biological functions and complex relationship with other organelles in a variety of dynamic physiological processes which necessitates efficient and controllable chemical tools. We herein report a photo-oxidation driven proximity labeling strategy to profile the mitochondrial proteome by light dependence in living cells with high spatiotemporal resolution. Taking advantage of organelle-localizable organic photoactivated probes generating reactive species and nucleophilic substrates for proximal protein oxidation and trapping, mitochondrial proteins were selectively labeled by spatially limited reactions in their native environment. Integration of photo-oxidation driven proximity labeling and quantitative proteomics facilitated the plotting of the mitochondrial proteome in which up to 310 mitochondrial proteins were identified with a specificity of 64% in HeLa cells. Furthermore, mitochondrial proteome dynamics was deciphered in drug resistant Huh7 and LPS stimulated HMC3 cells which were hard-to-transfect. A number of differential proteins were quantified which were intimately linked to critical processes and provided insights into the related molecular mechanisms of drug resistance and neuroinflammation in the perspective of mitochondria. The photo-oxidation driven proximity labeling strategy offers solid technical support to a highly precise proteomic platform in time and finer space for more knowledge of subcellular biology.

Segmented MS/MS acquisition of a1 ion-based strategy for in-depth proteome quantitation.

In-depth proteome quantitation is of great significance for understanding protein functions, advancing biological, medical, environmental and metabolic engineering research. Herein, benefiting from the high formation efficiencies and intensities of dimethyl-labeled a1 ions for accurate quantitation, we developed an in-depth a1 ion-based proteome quantitation method, named deep-APQ, by a sequential MS/MS acquisition of the high mass range for identification and the low mass range for a1 ion intensity extraction to increase quantitative protein number and sequence coverage. By the analysis of HeLa protein digests, our developed method showed deeper quantitative coverage than our previously reported a1 ion-based quantitation method without mass range segmentation and lower missing values than widely-used label-free quantitation method. It also exhibited excellent accuracy and precision within a 20-fold dynamic range. We further integrated a workflow combining the deep-APQ method with highly efficient sample preparation, high-pH and low-pH reversed-phase separation and high-field asymmetric waveform ion mobility spectrometry (FAIMS) to study E. coli proteome responses under the nutritional conditions of glucose and acetate. A total of 3447 proteins were quantified, representing 82% of protein-coding genes, with the average sequence coverage up to 40%, demonstrating the high coverage of quantitation results. We found that most of the quantified proteins related to chemotaxis were differentially expressed, including the low-abundance proteins such as tap, trg, aer, cheA and cheB, indicating that chemotaxis would play an important role for E. coli cell to survive from acetate toxicity. The above results demonstrated that the deep-APQ method is of great promising to achieve the deep-coverage proteome quantitation with high confidence.