Application of Machine Learning Techniques to Development of Emergency Medical Rapid Triage Prediction Models in Acute Care.

Given the critical and complex features of medical emergencies, it is essential to develop models that enable prompt and suitable clinical decision-making based on considerable information. Emergency nurses are responsible for categorizing and prioritizing injuries and illnesses on the frontlines of the emergency room. This study aims to create an Emergency Medical Rapid Triage and Prediction Assistance model using electronic medical records and machine learning techniques. Patient information was retrieved from the emergency department of a large regional teaching hospital in Taiwan, and five supervised learning techniques were used to construct classification models for predicting critical outcomes. Of these models, the model using logistic regression had superior prediction performance, with an F1 score of 0.861 and an area under the receiver operating characteristic curve of 0.855. The Emergency Medical Rapid Triage and Prediction Assistance model demonstrated superior performance in predicting intensive care and hospitalization outcomes compared with the Taiwan Triage and Acuity Scale and three clinical early warning tools. The proposed model has the potential to assist emergency nurses in executing challenging triage assessments and emergency teams in treating critically ill patients promptly, leading to improved clinical care and efficient utilization of medical resources.

REST-repressed lncRNA LINC01801 induces neuroendocrine differentiation in prostate cancer via transcriptional activation of autophagy.

The association between REST reduction and the development of neuroendocrine prostate cancer (NEPC), a novel drug-resistant and lethal variant of castration-resistant prostate cancer (CRPC), is well established. To better understand the mechanisms underlying this process, we aimed to identify REST-repressed long noncoding RNAs (lncRNAs) that promote neuroendocrine differentiation (NED), thus facilitating targeted therapy-induced resistance. In this study, we used data from REST knockdown RNA sequencing combined with siRNA screening to determine that LINC01801 was upregulated and played a crucial role in NED in prostate cancer (PCa). Using The Cancer Genome Atlas (TCGA) prostate adenocarcinoma database and CRPC samples collected in our laboratory, we demonstrated that LINC01801 expression is upregulated in NEPC. Functional experiments revealed that overexpression of LINC01801 had a slight stimulatory effect on the NED of LNCaP cells, while downregulation of LINC01801 significantly inhibited the induction of NED. Mechanistically, LINC01801 is transcriptionally repressed by REST, and transcriptomic analysis revealed that LINC01801 preferentially affects the autophagy pathway. LINC01801 was found to function as a competing endogenous RNA (ceRNA) to regulate the expression of autophagy-related genes by sponging hsa-miR-6889-3p in prostate cancer cells. In conclusion, our data expand the current knowledge of REST-induced NED and highlight the contribution of the REST-LINC01801-hsa-miR-6889-3p axis to autophagic induction, which may provide promising avenues for therapeutic opportunities.

Risk factors for hemorrhagic complications following robotic-assisted partial nephrectomy.

BACKGROUND: The aim of this study was to analyze the risk factors for hemorrhagic complications in patients who underwent robotic-assisted partial nephrectomy. METHODS: We retrospectively reviewed the records of 260 patients who underwent robotic-assisted partial nephrectomy. Hemorrhagic complications were defined as bleeding, hematoma, or arteriovenous fistula requiring hemostatic medication, blood transfusion, or therapeutic intervention. Hemorrhagic complications were graded according to the modified Clavien classification system, and the hemorrhagic complication group comprised only those complications with Clavien grade II or higher. Thereafter, we investigated the presence of any relevant association between perioperative factors and hemorrhagic complications. RESULTS: Of 260 patients included in the study, 32 (12.3%) had hemorrhagic complications. The postoperative hemoglobin level was significantly lower in the hemorrhagic complication group than in the group without complications. The hemorrhagic complication group had significantly more essential blood loss and a significantly longer length of hospital stay. In the univariate analysis, type 2 diabetes mellitus, Radius-scores tumor size as maximal diameter exophytic/endophytic properties of the tumor nearness of the deepest portion of the tumor to the collecting system or renal sinus anterior (a)/posterior (p) descriptor location relative to the polar line., sum of the renal size plus renal sinus involvement in the PADUA score is a simple anatomical system that can be used to predict the risk of surgical and medical perioperative complications in patients undergoing open NSS, prolonged console time (>180 minutes), prolonged warm ischemic time (>25 minutes), and method of pedicle control were statistically significant risk factors. In the multivariate logistic regression analysis, warm ischemic time >25 minutes was the only significant risk factor for hemorrhagic complications (odds ratio, 3.51; 95% confidence interval, 1.28-9.59; p = 0.01). CONCLUSION: Patients who undergo robotic-assisted partial nephrectomy with a warm ischemic time >25 minutes are significantly more likely to have hemorrhagic complications and should hence receive careful perioperative follow-up.

Risk factors for pharmacotherapy for storage symptoms after transurethral resection of the prostate in patients with benign prostatic hyperplasia.

OBJECTIVES: Postoperative persistence of storage symptoms after transurethral resection of the prostate (TURP) is bothersome, and evidence of its cause is sparse. We sought to analyze risk factors for using antimuscarinics or beta-3 agonists after TURP in benign prostatic hyperplasia (BPH) patients. METHODS: BPH patients who underwent TURP and were followed up for >6 months after surgery were retrospectively enrolled. Postoperative pharmacotherapy for storage symptoms was defined as the prescription of antimuscarinics or beta-3 agonists within 3 months after TURP for >3 months. Preoperative and perioperative variables were evaluated for their effect on the postoperative prescription of antimuscarinics or beta-3 agonists. RESULTS: Of the 376 patients, 45 (12.0%) received postoperative pharmacotherapy for storage symptoms. Patients who underwent bipolar TURP were significantly more likely to receive postoperative pharmacotherapy than those who underwent monopolar TURP (15.7% vs 6.9%; P = 0.01). Significantly more patients with intravesical prostatic protrusions >1 cm used postoperative pharmacotherapy than those with protrusions of ≤1 cm (14.4% vs 5.2% respectively; P = 0.02). Multivariate logistic regression analysis revealed age >75 years (odds ratio [OR] 3.04; 95% CI 1.29-7.16; P = 0.011), intravesical prostatic protrusion >1 cm (OR, 3.48; 95% CI, 1.32-9.15; P = 0.012), and bipolar transurethral resection (OR 4.25; 95% CI 1.53-11.80; P = 0.005) as significant risk factors for postoperative pharmacotherapy. CONCLUSIONS: Advanced age, intravesical prostatic protrusion, and bipolar TURP were significantly associated with postoperative pharmacotherapy for storage symptoms after TURP in BPH patients. Therefore, patients with these risk factors might be informed about the risk of postoperative storage symptoms that may require medications after TURP.

Proteomic characterization of arsenic and cadmium exposure in bladder cells.

RATIONALE: Accumulating evidence has linked prolonged exposure to heavy metals to cancer occurrence in the urinary system. However, the specific biological mechanisms responsible for the association of heavy metals with the unusually high incidence of upper tract urothelial carcinoma in Taiwan are complex and incompletely understood. METHODS: To elucidate the specific biological mechanism and identify molecular indicators of the unusually high association of upper tract urothelial carcinoma with heavy metal exposure, protein expression following the treatment of T24 human bladder carcinoma and RT4 human bladder papilloma cell line models with arsenic (As) and cadmium (Cd) was studied. Proteomic changes in these cell models were integrated with data from a human bladder cancer (BLCA) tissue proteome to identify possible protein indicators of heavy metal exposure. RESULTS: After mass spectrometry based proteomic analysis and verification by Western blotting procedures, we identified 66 proteins that were up-regulated and 92 proteins that were down-regulated in RT4 cell extracts after treatment with As or Cd. Some 52 proteins were up-regulated and 136 proteins were down-regulated in T24 cell extracts after treatment with Cd. We further confirmed that down-expression of the PML (promyelocytic leukemia) protein was sustained for at least 75 days after exposure of bladder cells to As. Dysregulation of these cellular proteins by As was associated with three biological pathways. Immunohistochemical analyses of paraffin-embedded BLCA tissue slides confirmed that PML protein expression was decreased in BLCA tumor cells compared with adjacent noncancerous epithelial cells. CONCLUSIONS: These data suggest that PML may play an important role in the pathogenesis of BLCA and may be an indicator of heavy metal exposure in bladder cells.

Development of biomarkers of genitourinary cancer using mass spectrometry-based clinical proteomics.

Prostate, bladder and kidney cancer are the three most common types of genitourinary cancer in the world. Of these, prostate and bladder cancers are within the top 10 most common cancers in men. Notably, kidney cancer causes no obvious symptoms in the early stages. To satisfy clinical-management requirements, researchers have developed numerous biomarkers by applying proteomic approaches using clinical serum, urine and tissue specimens, as well as cell and animal models. Through application of biomarker pipeline protocols, including discovery, verification and validation phases, and mass-spectrometric based proteomic platforms coupled with multiplexed quantification assays, these studies have led to recent rapid progress in this area. With improvements in mass-spectrometric based proteomic techniques, numerous promising biomarker candidates and marker panels for various clinical purposes have been proposed. Verification of novel protein biomarker candidates is very resource demanding (e.g. on the clinical and laboratory sides). With the support of national consortia, it is now possible to investigate the future clinical use of such biomarker strategies and assess their cost-effectiveness in personalized medicine.

Exploiting Honokiol-induced ER stress CHOP activation inhibits the growth and metastasis of melanoma by suppressing the MITF and ß-catenin pathways.

There is increasing global incidence of highly metastatic melanoma and therapeutic strategies like those focusing on the downstream beta-catenin/MITF axis of invading melanoma cells are urgently needed. Targeting endoplasmic reticulum (ER) stress can promote cancer cell death and inhibit epithelial mesenchymal transition (EMT) in metastatic tumors. This study aimed to determine if Honokiol could promote ER stress-dependent apoptosis and regulate metastatic melanoma. The therapeutic efficacy of Honokiol was assessed using the highly metastatic melanoma xenograft mouse model for peritoneal metastasis and evaluated by computed tomography imaging. The ER stress marker, Calpain-10, delineated a novel proteolytic cleavage enzyme, while CHOP/GADD153-regulated apoptosis was used for gene silencing to determine the role of the ß-catenin/MITF axis in melanoma cells. The results showed that Honokiol effectively decreased peritoneal dissemination and organ metastasis via ER stress activation and EMT marker inhibition. Knockdown Calpain-10 or CHOP/GADD153 blocked all of the biological effects in Honokiol-induced ß-catenin/MITF cleavage, ERSE or TCF/LEF luciferase activity, and ß-catenin kinase activity. These findings suggest that Honokiol can significantly thwart the progression of highly metastatic melanoma using the ß-catenin/MITF axis via prompt Calpain-10 and CHOP/GADD153 regulated cascades.

Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens.

Bladder cancer biomarkers currently approved by the Food and Drug Administration are insufficiently reliable for use in non-invasive clinical diagnosis. Verification/validation of numerous biomarker candidates for BC detection is a crucial bottleneck for novel biomarker development. A multiplexed liquid chromatography multiple-reaction-monitoring mass spectrometry assay of 122 proteins, including 118 up-regulated tissue proteins, two known bladder cancer biomarkers and two housekeeping gene products, was successfully established for protein quantification in clinical urine specimens. Quantification of 122 proteins was performed on a large cohort of urine specimens representing a variety of conditions, including 142 hernia, 126 bladder cancer, 67 hematuria, and 59 urinary tract infection samples. ANXA3 (annexin A3) and HSPE1 (heat shock protein family E member 1), which showed the highest detection frequency in bladder cancer samples, were selected for further validation. Western blotting showed that urinary ANXA3 and HSPE1 protein levels were higher in bladder cancer samples than in hernia samples, and enzyme-linked immunosorbent assays confirmed a higher urinary concentration of HSPE1 in bladder cancer than in hernia, hematuria and urinary tract infection. Immunohistochemical analyses showed significantly elevated levels of HSPE1 in tumor cells compared with non-cancerous bladder epithelial cells, suggesting that HSPE1 could be a useful tumor tissue marker for the specific detection of bladder cancer. Collectively, our findings provide valuable information for future validation of potential biomarkers for bladder cancer diagnosis.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research.

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer.

More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins-SLC3A2, STMN1, and TAGLN2-in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant overexpression in individual bladder cancer tissues and urine specimens, and thus represents a potential biomarker for noninvasive screening for bladder cancer. Our findings highlight the value of bladder tissue proteome in providing valuable information for future validation studies of potential biomarkers in urothelial carcinoma.

Enhancement of tumor initiation and expression of KCNMA1, MORF4L2 and ASPM genes in the adenocarcinoma of lung xenograft after vorinostat treatment.

Cancer stem cells (CSCs) are usually tolerant to chemotherapy and radiotherapy and associated with tumor relapse. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), is currently being used in clinical trials of lung cancer. However, SAHA facilitates the formation of induced pluripotent stem cells from somatic cells. We hypothesized that SAHA would mediate the CSCs properties and subsequently confer a more malignant phenotype in lung cancer. Transfected H1299 lung cancer cells, which stably expresses a triple fused reporter gene (DsRedm-Fluc-tTKsr39) under the control of CMV promoter was used to establish a xenograft mouse model. After the treatment of SAHA, H1299 cell line and tumor xenografts were sorted by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity. We found that SAHA could suppress the growth of xenografted H1299 tumors with decreased proportion of ALDHbr lung cancer cells indicating that SAHA may target CSCs. However, SAHA significantly enhanced the tumor initiating capacity and the expression of malignant genes such as KCNMA1, MORF4L2 and ASPM in the remaining living ALDHbr cells. These findings suggested that SAHA treatment created a more drug-resistant state in residual ALDHbr cells. The in vivo imaging technique may facilitate searching and characterization of CSCs.

Over-expression of cofilin-1 suppressed growth and invasion of cancer cells is associated with up-regulation of let-7 microRNA.

Cofilin-1, a non-muscle isoform of actin regulatory protein that belongs to the actin-depolymerizing factor (ADF)/cofilin family is known to affect cancer development. Previously, we found that over-expression of cofilin-1 suppressed the growth and invasion of human non-small cell lung cancer (NSCLC) cells in vitro. In this study, we further investigated whether over-expression of cofilin-1 can suppress tumor growth in vivo, and performed a microRNA array analysis to better understand whether specific microRNA would be involved in this event. The results showed that over-expression of cofilin-1 suppressed NSCLC tumor growth using the xenograft tumor model with the non-invasive reporter gene imaging modalities. Additionally, cell motility and invasion were significantly suppressed by over-expressed cofilin-1, and down-regulation of matrix metalloproteinase (MMPs) -1 and -3 was concomitantly detected. According to the microRNA array analysis, the let-7 family, particularly let-7b and let-7e, were apparently up-regulated among 248 microRNAs that were affected after over-expression of cofilin-1 up to 7 days. Knockdown of let-7b or let-7e using chemical locked nucleic acid (LNA) could recover the growth rate and the invasion of cofilin-1 over-expressing cells. Next, the expression of c-myc, LIN28 and Twist-1 proteins known to regulate let-7 were analyzed in cofilin-1 over-expressing cells, and Twist-1 was significantly suppressed under this condition. Up-regulation of let-7 microRNA by over-expressed cofilin-1 could be eliminated by co-transfected Twist-1 cDNA. Taken together, current data suggest that let-7 microRNA would be involved in over-expression of cofilin-1 mediated tumor suppression in vitro and in vivo.

Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics.

In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer. BIOLOGICAL SIGNIFICANCE: In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time.

Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity.

PURPOSE: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. MATERIALS AND METHODS: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. RESULTS: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. CONCLUSION: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.

Comparative and targeted proteomic analyses of urinary microparticles from bladder cancer and hernia patients.

Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary microparticles are an attractive material for noninvasive bladder cancer biomarker discovery. In this study, we applied isotopic dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to discover bladder cancer biomarkers in urinary microparticles isolated from hernia (control) and bladder cancer patients. This approach identified 2964 proteins based on more than two distinct peptides, of which 2058 had not previously been reported as constituents of human urine exosomes/microparticles. A total of 107 differentially expressed proteins were identified as candidate biomarkers. Differences in the concentrations of 29 proteins (41 signature peptides) were precisely quantified by LC-MRM/MS in 48 urine samples of bladder cancer, hernia, and urinary tract infection/hematuria. Concentrations of 24 proteins changed significantly (p<0.05) between bladder cancer (n=28) and hernia (n=12), with area-under-the-curve values ranging from 0.702 to 0.896. Finally, we quantified tumor-associated calcium-signal transducer 2 (TACSTD2) in raw urine specimens (n=221) using a commercial ELISA and confirmed its potential value for diagnosis of bladder cancer. Our study reveals a strong association of TACSTD2 with bladder cancer and highlights the potential of human urinary microparticles in the noninvasive diagnosis of bladder cancer.

Proline in transmembrane domain of type II protein DPP-IV governs its translocation behavior through endoplasmic reticulum.

A transmembrane domain (TMD) at the N-terminus of a membrane protein is a signal sequence that targets the protein to the endoplasmic reticulum (ER) membrane. Proline is found more frequently in TM helices compared to water-soluble helices. To investigate the effects of proline on protein translocation and integration in mammalian cells, we made proline substitutions throughout the TMD of dipeptidyl peptidase IV, a type II membrane protease with a single TMD at its N-terminus. The proteins were expressed and their capacities for targeting and integrating into the membrane were measured in both mammalian cells and in vitro translation systems. Three proline substitutions in the central region of the TMD resulted in various defects in membrane targeting and/or integration. The replacement of proline with other amino acids of similar hydrophobicity rescued both the translocation and anchoring defects of all three proline mutants, indicating that conformational change caused by proline is a determining factor. Increasing hydrophobicity of the TMD by replacing other residues with more hydrophobic residues also effectively reversed the translocation and integration defects. Intriguingly, increasing hydrophobicity at the C-terminal end of the TMD rescued much more effectively than it did at the N-terminal end. Thus, the effect of proline on translocation and integration of the TMD is not determined solely by its conformation and hydrophobicity, but also by the location of proline in the TMD, the location of highly hydrophobic residues, and the relative position of the proline to other proline residues in the TMD.

The dimeric transmembrane domain of prolyl dipeptidase DPP-IV contributes to its quaternary structure and enzymatic activities.

Dipeptidyl peptidase IV (DPP-IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP-IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP-IV promotes DPP-IV dimerization and rescues monomeric DPP-IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP-IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site-directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP-IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline-containing TMs. Thus, the proline-dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the extracellular active site. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP-IV. Optimal enzymatic activity of DPP-IV requires an optimal interaction of all three dimer interfaces, including its TM.

Supraphysiological thermal injury in different human bladder carcinoma cell lines.

Depending on the duration of exposure to supraphysiological temperatures, cellular proteins and organelles can suffer from structural alternations and irreversible denaturation, which may induce cell death. The thermotolerance of three human urinary bladder carcinoma cell lines, TSGH-8301, J82 and TCC-SUP (cytological grade 2, 3 and 4, respectively), was investigated in the present study. A home-made heating stage was used to provide a constant temperature for different cell lines of bladder carcinoma. The experimental data showed that the TCC-SUP and TSGH-8301 cells exhibited the lowest and highest thermotolerances, respectively, while J82 cells were intermediate. Moreover, the differences in the thermotolerances for the TSGH-8301 and J82 cells are significant when the supraphysiological temperature is higher than 43 degrees C. As for TSGH-8301 and TCC-SUP cells, the thermotolerances are significantly different for all of the thermal treatments tested. Furthermore, the thermotolerances of J82 and TCC-SUP are significantly different when the cells are exposed to a temperature less than 50 degrees C for longer than 2 min. Overall, the results suggest that the high cytological grade of the cell line of bladder cancer exhibits a low thermotolerance. The kinematic parameters of the activation energy and frequency factor for bladder cancer cell lines with different cytological grades were also quantitatively evaluated in this work.

Regulated expression of cofilin and the consequent regulation of p27(kip1) are essential for G(1) phase progression.

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin upregulated in G(1) phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G(1) cells. To investigate the role of cofilin in the control of G(1) phase progression, a tet-on gene expression system was introduced to overexpress different concentrations of cofilin in cells. The results showed that G(1) phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G(1) phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27(kip1) was the primary molecule induced by overexpressed cofilin in a time and dose dependent manner. Upregulated p27(kip1) repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27(kip1) expression in the cofilin overexpressing cells released the G(1) phase arrest. Furthermore, we found that overexpression of cofilin led to induction of p27(kip1) gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27(kip1) mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27(kip1) protein for ubiquitinyl-proteasomal mediated degradation was also involved in upregulation of p27(kip1). These data suggest that cofilin expression and its regulation of p27(kip1) expression is important for the control of G(1) phase progression.