Adjuvant treatment strategy evolution and risk stratification for hormone receptor-positive, human epidermal growth factor receptor-2 negative early breast cancer in China.

BACKGROUND: Patients with hormone receptor-positive (HR+), human epidermal growth factor receptor-2 negative (HER2-) early breast cancer (EBC) with high-risk clinicopathological features face an increased risk of recurrence. This study explored the evolving treatment landscape and clinical outcomes in patients with EBC using a nationwide database. PATIENTS AND METHODS: The study cohort comprised HR+/HER2-, stages 1-3, patients with EBC who underwent surgery and received adjuvant endocrine therapy (AET) from January 2013 to March 2021. High-risk patients were defined by ≥4 positive axillary lymph nodes, or 1-3 positive lymph node(s) with at least one high-risk feature (histologic grade 3, tumor size ≥5 cm, or Ki-67 ≥20%). A low-risk cohort included patients not meeting the criteria. Survival analysis was conducted with a cutoff of September 2021. RESULTS: The study included 4088 eligible patients (1310 high-risk patients and 2778 low-risk patients). High-risk patients were more likely to receive adjuvant chemotherapy and radiotherapy compared to low-risk patients. From 2013 to 2021, an increasing proportion of patients received aromatase inhibitors and ovarian function suppression as part of their AET. The 2-, 5-, and 7-year invasive disease-free survival for high-risk cohort were 90.67%, 75.26%, and 57.10%, respectively, these rates were notably higher for low-risk cohort at 97.14%, 89.85%, and 84.83%. High-risk patients demonstrated a higher risk of recurrence or death compared with low-risk patients (hazard ratio, 2.38; 95% CI, 1.82-3.12). CONCLUSION: In the setting of standard or even intensive AET, patients with EBC with high-risk features still present high recurrence risk, highlighting the urgent need for innovative adjuvant treatment strategies.

PITPNC1 Suppress CD8+ T cell immune function and promote radioresistance in rectal cancer by modulating FASN/CD155.

BACKGROUND: Radioresistance is a primary factor contributing to the failure of rectal cancer treatment. Immune suppression plays a significant role in the development of radioresistance. We have investigated the potential role of phosphatidylinositol transfer protein cytoplasmic 1 (PITPNC1) in regulating immune suppression associated with radioresistance. METHODS: To elucidate the mechanisms by which PITPNC1 influences radioresistance, we established HT29, SW480, and MC38 radioresistant cell lines. The relationship between radioresistance and changes in the proportion of immune cells was verified through subcutaneous tumor models and flow cytometry. Changes in the expression levels of PITPNC1, FASN, and CD155 were determined using immunohistochemistry and western blotting techniques. The interplay between these proteins was investigated using immunofluorescence co-localization and immunoprecipitation assays. Additionally, siRNA and lentivirus-mediated gene knockdown or overexpression, as well as co-culture of tumor cells with PBMCs or CD8+ T cells and establishment of stable transgenic cell lines in vivo, were employed to validate the impact of the PITPNC1/FASN/CD155 pathway on CD8+ T cell immune function. RESULTS: Under irradiation, the apoptosis rate and expression of apoptosis-related proteins in radioresistant colorectal cancer cell lines were significantly decreased, while the cell proliferation rate increased. In radioresistant tumor-bearing mice, the proportion of CD8+ T cells and IFN-γ production within immune cells decreased. Immunohistochemical analysis of human and animal tissue specimens resistant to radiotherapy showed a significant increase in the expression levels of PITPNC1, FASN, and CD155. Gene knockdown and rescue experiments demonstrated that PITPNC1 can regulate the expression of CD155 on the surface of tumor cells through FASN. In addition, co-culture experiments and in vivo tumor-bearing experiments have shown that silencing PITPNC1 can inhibit FASN/CD155, enhance CD8+ T cell immune function, promote colorectal cancer cell death, and ultimately reduce radioresistance in tumor-bearing models. CONCLUSIONS: PITPNC1 regulates the expression of CD155 through FASN, inhibits CD8+ T cell immune function, and promotes radioresistance in rectal cancer.

Clinical activity and safety of sintilimab, bevacizumab, and TMZ in patients with recurrent glioblastoma.

PURPOSE: There are limited and no standard therapies for recurrent glioblastoma. We herein report the antitumour activity and safety of sintilimab, bevacizumab and temozolomide (TMZ) in recurrent glioblastoma. METHODS: We retrospectively analysed eight patients with recurrent glioblastoma treated with sintilimab (200 mg) every three weeks + bevacizumab (10 mg/kg) every three weeks + TMZ (200 mg/m²orally) (5 days orally every 28 days for a total of four weeks). The primary objective was investigator-assessed median progression-free survival(mPFS). Secondary objectives were to assess the 6-month PFS, objective response rate (ORR) and duration of response (DOR) accroding to RANO criteria. RESULTS: The mPFS time for 8 patients was 3.340 months (95% CI: 2.217-4.463), The longest PFS was close to 9 months. Five patients were assessed to have achieved partial response (PR), with an overall remission rate of 62.5%, Four patients experienced a change in tumour volume at the best response time of greater than 60% shrinkage from baseline, and one patient remained progression free upon review, with a DOR of more than 6.57 months. The 6-month PFS was 25% (95% CI: 5.0-55.0%). Three patients had a treatment-related adverse events, though no grade 4 or 5 adverse events occurred. CONCLUSION: In this small retrospective study, the combination regimen of sintilimab, bevacizumab and TMZ showed promising antitumour activity in treatment of recurrent glioblastoma, with a good objective remission rate.

Isolation and genome-wide analysis of the novel Acinetobacter baumannii bacteriophage vB_AbaM_AB3P2.

A lytic Acinetobacter baumannii phage, isolate vB_AbaM_AB3P2, was isolated from a sewage treatment plant in China. A. baumannii phage vB_AbaM_AB3P2 has a dsDNA genome that is 44,824 bp in length with a G + C content of 37.75%. Ninety-six open reading frames were identified, and no genes for antibiotic resistance or virulence factors were found. Genomic and phylogenetic analysis of this phage revealed that it represents a new species in the genus Obolenskvirus. Phage vB_AbaM_AB3P2 has a short latent period (10 min) and high stability at 30-70°C and pH 2-10 and is potentially useful for controlling multi-drug-resistant A. baumannii.

Unraveling the unfolded protein response signature: implications for tumor immune microenvironment heterogeneity and clinical prognosis in stomach cancer.

BACKGROUND: Stomach cancer is a leading cause of cancer-related deaths globally due to its high grade and poor response to treatment. Understanding the molecular network driving the rapid progression of stomach cancer is crucial for improving patient outcomes. METHODS: This study aimed to investigate the role of unfolded protein response (UPR) related genes in stomach cancer and their potential as prognostic biomarkers. RNA expression data and clinical follow-up information were obtained from the TCGA and GEO databases. An unsupervised clustering algorithm was used to identify UPR genomic subtypes in stomach cancer. Functional enrichment analysis, immune landscape analysis, and chemotherapy benefit prediction were conducted for each subtype. A prognostic model based on UPR-related genes was developed and validated using LASSO-Cox regression, and a multivariate nomogram was created. Key gene expression analyses in pan-cancer and in vitro experiments were performed to further investigate the role of the identified genes in cancer progression. RESULTS: A total of 375 stomach cancer patients were included in this study. Analysis of 113 UPR-related genes revealed their close functional correlation and significant enrichment in protein modification, transport, and RNA degradation pathways. Unsupervised clustering identified two molecular subtypes with significant differences in prognosis and gene expression profiles. Immune landscape analysis showed that UPR may influence the composition of the tumor immune microenvironment. Chemotherapy sensitivity analysis indicated that patients in the C2 molecular subtype were more responsive to chemotherapy compared to those in the C1 molecular subtype. A prognostic signature consisting of seven UPR-related genes was constructed and validated, and an independent prognostic nomogram was developed. The gene IGFBP1, which had the highest weight coefficient in the prognostic signature, was found to promote the malignant phenotype of stomach cancer cells, suggesting its potential as a therapeutic target. CONCLUSIONS: The study developed a UPR-related gene classifier and risk signature for predicting survival in stomach cancer, identifying IGFBP1 as a key factor promoting the disease's malignancy and a potential therapeutic target. IGFBP1's role in enhancing cancer cell adaptation to endoplasmic reticulum stress suggests its importance in stomach cancer prognosis and treatment.

Cdc42-driven endosomal cholesterol transport promotes collateral resistance in HER2-positive gastric cancer.

Resistance to trastuzumab and the poor efficacy of subsequent chemotherapy have become major challenges for HER2-positive gastric cancer (GC). As resistance evolves, tumor cells may acquire a new drug susceptibility profile, profoundly impacting the subsequent treatment selection and patient survival. However, the interplay between trastuzumab and other types of drugs in HER2-positive GC remains elusive. In our study, we utilized resistant cell lines and tissue specimens to map the drug susceptibility profile of trastuzumab-resistant GC, discovering that resistance to trastuzumab induces collateral resistance to commonly used chemotherapeutic agents. Additionally, patients with collateral resistance distinguished by a 13-gene scoring model in HER2-positive GC cohorts are predicted to have a poor prognosis and may be sensitive to cholesterol-lowering drugs. Mechanistically, endosomal cholesterol transport is further confirmed to enrich cholesterol in the plasma membrane, contributing to collateral resistance through the Hedgehog-ABCB1 axis. As a driver for cholesterol, Cdc42 is activated by the formation of the NPC1-TßRI-Cdc42 complex to facilitate endosomal cholesterol transport. We demonstrated that inhibiting Cdc42 activation with ZCL278 reduces cholesterol levels in the plasma membrane and reverses collateral resistance between trastuzumab and chemotherapy in vitro and in vivo. Collectively, our findings verify the phenomena and mechanism of collateral resistance between trastuzumab and chemotherapy, and propose a potential therapeutic target and strategy in the second-line treatment for trastuzumab-resistant HER2-positive GC.

Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation.

Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of nonphosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment from 5 mL of 1 fmol/µL phosphoprotein digests and concentration into small-volume (tens of microliters) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer's disease.

Co-occurrence of blaNDM-1, rmtC, and mcr-9 in multidrug-resistant Enterobacter kobei strain isolated from an infant with urinary tract infection.

OBJECTIVES: The co-emergence of mcr and carbapenem resistance genes in Gram-negative bacteria is a serious problem. This study aims to clarify the genetic characteristic of one novel multidrug-resistant Enterobacter kobei EC1382 with mcr-9 causing urinary tract inflammation in an infant. METHODS: Antimicrobial drug susceptibility testing was performed for this isolate using the broth microdilution method. Whole-genome sequencing was performed using the Illumina PacBio RS II platform and HiSeq platform, and the antimicrobial resistance genes, mobile elements, and plasmid replicon types were identified. Conjugation analysis was performed using Escherichia coli C600 as recipients. RESULTS: Enterobacter kobei EC1382 was resistant to carbapenem, aminoglycoside, and cephalosporin. Twenty-five antimicrobial resistance genes were identified, including genes conferring resistance to carbapenem (blaNDM-1), colistin (mcr-9), and aminoglycosides (rmtC). The blaNDM-1 gene, accompanied by bleMBL and rmtC located downstream of an ISCR14 element, was detected in the IncFII(Yp) type plasmid pEC1382-2. Interestingly, although E. kobei EC1382 was susceptible to colistin, it had three identical mcr-9 genes (two in the chromosome and one in the IncHI2-type plasmid pEC1382-1). The backbone (∼12.2-kb genetic fragment) of these mcr-9 (flanked by IS903B and IS481-IS26) regions were conserved in this strain, and they were found to be present in various bacteria as three types, implying a silent distribution. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate the coexistence of blaNDM-1, rmtC, and mcr-9 in E. kobei. The silent prevalence of mcr-9 in bacteria may be a threat to public health.

Effect and mechanism of total ginsenosides repairing SDS­induced Drosophila enteritis model based on MAPK pathway.

Inflammatory bowel disease (IBD) is a chronic recurrent gastrointestinal disease that seriously endangers human and animal health. Although the etiology of IBD is complex and the pathogenesis is not well understood, studies have found that genetic predisposition, diet and intestinal flora disorders are the main risk factors for IBD. The potential biological mechanism of total ginsenosides (TGGR) in the treatment of IBD remains to be elucidated. Surgery is still the main strategy for the treatment of IBD, due to the relatively high side effects of related drugs and the easy development of drug resistance. The purpose of the present study was to evaluate the efficacy of TGGR and explore the effect of TGGR on the intestinal inflammation induced by sodium dodecyl sulfate (SDS) in Drosophila and to initially explain the improvement effect and mechanism of TGGR on Drosophila enteritis by analyzing the levels of Drosophila-related proteins. During the experiment, the survival rate, climb index and abdominal characteristics of the Drosophila was recorded. Intestinal samples of Drosophila were collected for analysis of intestinal melanoma. The oxidative stress related indexes of catalase, superoxide dismutase and malondialdehyde were determined by spectrophotometry. Western blotting detected the expression of signal pathway-related factors. The effects of TGGR on growth indices, tissue indices, biochemical indices, signal pathway transduction and related mechanisms of SDS-induced Drosophila enteritis model were studied. The results showed that TGGR could repair SDS-induced enteritis of Drosophila through MAPK signaling pathway, improve survival rate and climbing ability and repair intestinal damage and oxidative stress damage. The results suggested that TGGR has potential application value in the treatment of IBD and its mechanism is related to the downregulation of phosphorylated (p)-JNK/p-ERK levels, which provides a basis for drug research in the treatment of IBD.

Gravity-driven rattan-based catalytic filter for rapid and highly efficient organic pollutant removal.

Metal organic frameworks hold great promise as heterogeneous catalysts in sulfate radical (SO4∙-) based advanced oxidation. However, the aggregation of powdered MOF crystals and the complicated recovery procedure largely hinder their large-scale practical applications. It is important to develop eco-friendly and adaptable substrate-immobilized metal organic frameworks. Based on the hierarchical pore structure of the rattan, gravity-driven metal organic frameworks loaded rattan-based catalytic filter was designed to degrade organic pollutants by activating PMS at high liquid fluxes. Inspired by the water transportation of rattan, ZIF-67 was in-situ grown uniformly on the rattan channels inner surface using the continuous flow method. The intrinsically aligned microchannels in the vascular bundles of rattan acted as reaction compartments for the immobilization and stabilization of ZIF-67. Furthermore, the rattan-based catalytic filter exhibited excellent gravity-driven catalytic activity (up to 100 % treatment efficiency for a water flux of 10173.6 L·m-2·h-1), recyclability, and stability of organic pollutant degradation. After ten cycles, the TOC removal of ZIF-67@rattan was 69.34 %, maintaining a stable mineralisation capacity for pollutants. The inhibitory effect of the micro-channel promoted the interaction between active groups and contaminants, increasing the degradation efficiency and improving the stability of the composite. The design of a gravity-driven rattan-based catalytic filter for wastewater treatment provides an effective strategy for developing renewable and continuous catalytic systems.

Ginsenoside Rd, a natural production for attenuating fibrogenesis and inflammation in hepatic fibrosis by regulating the ERRα-mediated P2X7r pathway.

Ginseng, when used as a food and nutritional supplement, has the ability to regulate human immunity. Here, the potential anti-hepatic fibrosis effect of ginsenoside Rd (Rd), one of the protopanaxadiol types of ginsenoside, was investigated. We established a hepatic fibrosis model using intraperitoneal injection of thioacetamide (TAA) for five weeks in mice. In addition, an in vitro model was established by using TGF-ß to activate hepatic stellate cells (HSCs), treated with Rd and an estrogen-related receptor α (ERRα) inhibitor (XCT-790). The ERRα knockdown (shRNA-ERRα) of the primary mouse hepatocytes was used to establish hepatocyte injury by TGF-ß, and they were then incubated in Rd. The Rd significantly alleviated the histopathological changes, and reduced the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. The Rd could upregulate the ERRα and downregulate the fibrosis markers in the livers of mice. In TAA-induced mice, the Rd inhibited the purinergic ligand-gated ion channel 7 receptor (P2X7r)-mediated NLRP3 inflammasome activation, consequently reversing the liver inflammatory response. The Rd significantly increased the expression of ERRα and suppressed the extracellular matrix (ECM) in the HSCs or primary hepatocytes. The Rd significantly decreased the P2X7r-mediated NLRP3 inflammasome activation, consequently reversing the inflammatory response, including the production of IL-1ß, IL-23 in the activated HSCs and primary hepatocytes. The Rd could ameliorate the damage of the hepatocytes and further inhibit the entry of IL-1ß and IL-18 into the extracellular matrix. The Rd reduced the inflammatory reaction by regulating the ERRα-P2X7r signaling pathway while suppressing the fibrogenesis, which suggests that the Rd can serve as a novel dietary supplement approach to combat hepatic fibrosis.

Efficacy of everolimus-based therapy in advanced triple-positive breast cancer: Experience from three cancer centers in China.

BACKGROUND: Triple-positive breast cancer (TPBC) is highly invasive and lacks well-established treatment strategies, especially in patients with advanced stage disease. This study aimed to explore the efficacy of everolimus in patients with metastatic TPBC (mTPBC) in a multicenter real-world setting. METHODS: A total of 2518 cancer patients who received everolimus-based therapy were enrolled from three cancer institutes in China from 2014 to 2022. Their clinicopathological characteristics were collected from medical records. The indicators for the efficacy of everolimus were progression-free survival (PFS), objective response rate (ORR) and clinical benefit rate (CBR). RESULTS: We collected 79 HER2-enriched patients that were treated with everolimus-based therapy, 43 of whom were mTPBC. The most commonly used therapeutic combinations was everolimus plus endocrine therapy (18/43, 41.9%). Among all combinations, everolimus plus chemotherapy plus trastuzumab developed the longest PFS of 10.9 months (95% CI: 1.5-20.3). Seventeen patients (32.6%) with mTPBC received everolimus as frontline treatment (1 L/2 L/3 L, FL), and 26 patients (67.4%) as backline treatment (>3 L, BL). Among all the population, the median PFS for everolimus was 4.5 months (range: 3.0-6.0), ORR was 30.2%, and CBR was 48.8%. PFSFL of 10.9 months was significantly longer than 4.0 months for PFSBL (p = 0.003, HR = 0.31, 95% CI: 0.14-0.67). ORRFL was 41.2%, showing no significance compared to ORRBL of 23.1% (one-sided p = 0.11). CBRFL was observed better of 76.5% versus CBRBL of 46.2% (one-sided p = 0.026). CONCLUSION: Everolimus as frontline treatment achieves clinical benefits for Chinese patients with mTPBC, which may provide some references for the management of Chinese mTPBC patients.

FBXO7 Confers Mesenchymal Properties and Chemoresistance in Glioblastoma by Controlling Rbfox2-Mediated Alternative Splicing.

Mesenchymal glioblastoma (GBM) is highly resistant to radio-and chemotherapy and correlates with worse survival outcomes in GBM patients; however, the underlying mechanism determining the mesenchymal phenotype remains largely unclear. Herein, it is revealed that FBXO7, a substrate-recognition component of the SCF complex implicated in the pathogenesis of Parkinson's disease, confers mesenchymal properties and chemoresistance in GBM by controlling Rbfox2-mediated alternative splicing. Specifically, FBXO7 ubiquitinates Rbfox2 Lys249 through K63-linked ubiquitin chains upon arginine dimethylation at Arg341 and Arg441 by PRMT5, leading to Rbfox2 stabilization. FBXO7 controls Rbfox2-mediated splicing of mesenchymal genes, including FoxM1, Mta1, and Postn. FBXO7-induced exon Va inclusion of FoxM1 promotes FoxM1 phosphorylation by MEK1 and nuclear translocation, thereby upregulates CD44, CD9, and ID1 levels, resulting in GBM stem cell self-renewal and mesenchymal transformation. Moreover, FBXO7 is stabilized by temozolomide, and FBXO7 depletion sensitizes tumor xenografts in mice to chemotherapy. The findings demonstrate that the FBXO7-Rbfox2 axis-mediated splicing contributes to mesenchymal transformation and tumorigenesis, and targeting FBXO7 represents a potential strategy for GBM treatment.

Limited proteolysis via millisecond digestions in protease-modified membranes.

Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm(3)) and small pore diameters in the membrane lead to digestion in <1 s, the low membrane thickness (170 µm) affords control over residence times at the millisecond level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kDa) that increase the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion). With this approach, we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, Root Hair Defective 3 (RHD3) and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.

Recent Advances in Phage-Based Therapeutics for Multi-Drug Resistant Acinetobacter baumannii.

Acinetobacter baumannii is an important opportunistic pathogen common in clinical infections. Phage therapy become a hot research field worldwide again after the post-antibiotic era. This review summarizes the important progress of phage treatments for A. baumannii in the last five years, and focus on the new interesting advances including the combination of phage and other substances (like photosensitizer), and the phage encapsulation (by microparticle, hydrogel) in delivery. We also discuss the remaining challenges and promising directions for phage-based therapy of A. baumannii infection in the future, and the innovative combination of materials in this area may be one promising direction.

Remodeling Chondroitin-6-Sulfate-Mediated Immune Exclusion Enhances Anti-PD-1 Response in Colorectal Cancer with Microsatellite Stability.

Metastatic microsatellite-stable (MSS) colorectal cancer rarely responds to immune checkpoint inhibitors (ICI). Metabolism heterogeneity in the tumor microenvironment (TME) presents obstacles to antitumor immune response. Combining transcriptome (The Cancer Genome Atlas MSS colorectal cancer, n = 383) and digital pathology (n = 96) analysis, we demonstrated a stroma metabolism-immune excluded subtype with poor prognosis in MSS colorectal cancer, which could be attributed to interaction between chondroitin-6-sulfate (C-6-S) metabolites and M2 macrophages, forming the "exclusion barrier" in the invasive margin. Furthermore, C-6-S derived from cancer-associated fibroblasts promoted co-nuclear translocation of pSTAT3 and GLI1, activating the JAK/STAT3 and Hedgehog pathways. In vivo experiments with C-6-S-targeted strategies decreased M2 macrophages and reprogrammed the immunosuppressive TME, leading to enhanced response to anti-PD-1 in MSS colorectal cancer. Therefore, C-6-S-induced immune exclusion represents an "immunometabolic checkpoint" that can be exploited for the application of combination strategies in MSS colorectal cancer ICI treatment.

Identification of p65-associated phosphoproteins by mass spectrometry after on-plate phosphopeptide enrichment using polymer-oxotitanium films.

Stimuli-induced protein phosphorylation plays a vital role in signal transduction and transcriptional activities in eukaryotic cells. This work aims to develop analysis techniques that rapidly detect stimulus-specific intracellular protein phosphorylation and association, with specific emphasis on identifying phosphoproteins associated with p65, a nuclear regulatory factor. The analytical strategy includes immunoprecipitation of the target protein along with its associated proteins, tryptic digestion directly on the antibody beads, on-plate phosphopeptide enrichment for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) to identify phosphopeptides and phosphorylation sites. Enrichment of the phosphopeptides from the tryptic digest occurs on a polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) and consumes as little as 1 microL of digest solution. The Au-P-oxoTi wafers can capture both mono- and multiphosphorylated peptides from model protein digests containing high concentrations of nonphosphopeptides, urea, and salts. When combined with MALDI-MS/MS, the enrichment technique reveals nine phosphopeptides from p65-associated proteins immunoprecipitated from human acute monocytic leukemia (THP-1) cell nuclear extracts. Semiquantitative MALDI-MS shows that the levels of these proteins increase dramatically after treatment with tumor necrosis factor (TNF)-alpha. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.

Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion.

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.

PITPNC1 fuels radioresistance of rectal cancer by inhibiting reactive oxygen species production.

BACKGROUND: Neoadjuvant radiotherapy is a commonly used method for the current standard-of-care for most patients with rectal cancer, when the effects of radioresistance are limited. The phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1), a lipid-metabolism-related gene, has previously been proved to manifest pro-cancer effects in multiple types of cancer. However, whether PITPNC1 plays a role for developing radioresistance in rectal cancer patients is still unknown. Therefore, this study aims to investigate the role of PITPNC1 in rectal cancer radioresistance. METHODS: Patient-derived tissue were used to detect the difference in the expression level of PITPNC1 between radioresistant and radiosensitive patients. Bioinformatic analyses of high-throughput gene expression data were applied to uncover the correlations between PITPNC1 level and oxidative stress. Two rectal cancer cell lines, SW620, and HCT116, were selected in vitro to investigate the effect of PITPNC1 on radioresistance, reactive oxygen species (ROS) generation, apoptosis, and proliferation in rectal cancer. RESULTS: PITPNC1 is highly expressed in radioresistant patient-derived rectal cancer tissues compared to radiosensitive tissue; therefore, PITPNC1 inhibits the generation of ROS and improves the extent of radioresistance of rectal cancer cell lines and then inhibits apoptosis. Knocking down PITPNC1 facilitates the production of ROS while application of the ROS scavenger, N-acetyl-L-cysteine (NAC), could reverse this effect. CONCLUSIONS: PITPNC1 fuels radioresistance of rectal cancer via the inhibition of ROS generation.

Shear stress activates ATOH8 via autocrine VEGF promoting glycolysis dependent-survival of colorectal cancer cells in the circulation.

BACKGROUND: Metastasis and recurrence, wherein circulating tumour cells (CTCs) play an important role, are the leading causes of death in colorectal cancer (CRC). Metastasis-initiating CTCs manage to maintain intravascular survival under anoikis, immune attack, and importantly shear stress; however, the underlying mechanisms remain poorly understood. METHODS: In view of the scarcity of CTCs in the bloodstream, suspended colorectal cancer cells were flowed into the cyclic laminar shear stress (LSS) according to previous studies. Then, we detected these suspended cells with a CK8+/CD45-/DAPI+ phenotype and named them mimic circulating tumour cells (m-CTCs) for subsequent CTCs related researches. Quantitative polymerase chain reaction, western blotting, and immunofluorescence were utilised to analyse gene expression change of m-CTCs sensitive to LSS stimulation. Additionally, we examined atonal bHLH transcription factor 8 (ATOH8) expressions in CTCs among 156 CRC patients and mice by fluorescence in situ hybridisation and flow cytometry. The pro-metabolic and pro-survival functions of ATOH8 were determined by glycolysis assay, live/dead cell vitality assay, anoikis assay, and immunohistochemistry. Further, the concrete up-and-down mechanisms of m-CTC survival promotion by ATOH8 were explored. RESULTS: The m-CTCs actively responded to LSS by triggering the expression of ATOH8, a fluid mechanosensor, with executive roles in intravascular survival and metabolism plasticity. Specifically, ATOH8 was upregulated via activation of VEGFR2/AKT signalling pathway mediated by LSS induced VEGF release. ATOH8 then transcriptionally activated HK2-mediated glycolysis, thus promoting the intravascular survival of colorectal cancer cells in the circulation. CONCLUSIONS: This study elucidates a novel mechanism that an LSS triggered VEGF-VEGFR2-AKT-ATOH8 signal axis mediates m-CTCs survival, thus providing a potential target for the prevention and treatment of hematogenous metastasis in CRC.