ProFun: A web server for functional enrichment analysis of parasitic protozoan genes.

BACKGROUND: The initial step to interpreting putative biological functions from comparative multi-omics studies usually starts from a differential expressed gene list followed by functional enrichment analysis (FEA). However, most FEA packages are designed exclusively for humans and model organisms. Although parasitic protozoan is the most important pathogen in the tropics, no FEA package is available for protozoan functional (ProFun) enrichment analysis. To speed up comparative multi-omics research on parasitic protozoans, we constructed ProFun, a web-based, user-friendly platform for the research community. METHODS: ProFun utilizes the Docker container, ShinyProxy, and R Shiny to construct a scalable web service with load-balancing infrastructure. We have integrated a series of visual analytic functions, in-house scripts, and custom-made annotation packages to create three analytical modules for 40 protozoan species: (1) Gene Overlaps; (2) Over-representation Analysis (ORA); (3) Gene Set Enrichment Analysis (GSEA). RESULTS: We have established ProFun, a web server for functional enrichment analysis of differentially expressed genes. FEA becomes as simple as pasting a list of gene IDs into the textbox of our website. Users can customize enrichment parameters and results with just one click. The intuitive web interface and publication-ready charts enable users to reveal meaningful biological events and pinpoint potential targets for further studies. CONCLUSION: ProFun is the first web application that enables gene functional enrichment analysis of parasitic protozoans. In addition to supporting FEA analysis, ProFun also allows the comparison of FEA results across complicated experimental designs. ProFun is freely available at http://dalek.cgu.edu.tw:8080/app/profun.

The core exosome proteome of Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is parasitic protozoan that causes human urogenital infections. Accumulated reports indicated that exosomes released by this parasite play a crucial role in transmitting information and substances between cells during host-parasite interactions. Current knowledge on the protein contents in T. vaginalis exosome is mainly generated from three previous studies that used different T. vaginalis isolates as an experimental model. Whether T. vaginalis exosomes comprise a common set of proteins (core exosome proteome) is still unclear. METHODS: To explore the core exosome proteome in T. vaginalis, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the contents of sucrose ultracentrifugation-enriched exosome and supernatant fractions isolated from six isolates. RESULTS: Transmission electron microscopy (TEM) confirmed the presence of exosomes in the enriched fraction. Proteomic analysis identified a total of 1870 proteins from exosomal extracts. There were 1207 exosomal-specific proteins after excluding 436 'non-core exosomal proteins'. Among these, 72 common exosomal-specific proteins were expressed in all six isolates. Compared with three published T. vaginalis exosome proteome datasets, we identified 16 core exosomal-specific proteins. These core exosomal-specific proteins included tetraspanin (TvTSP1), the classical exosome marker, and proteins mainly involved in catalytic activity and binding such as ribosomal proteins, ras-associated binding (Rab) proteins, and heterotrimeric G proteins. CONCLUSIONS: Our study highlighted the importance of using supernatant fraction from exosomal extract as a control to eliminate 'non-core exosomal proteins'. We compiled a reference core exosome proteome of T. vaginalis, which is essential for developing a fundamental understanding of exosome-mediated cell communication and host-parasite interaction.

A Swiss-Roll-Type Methanol Mini-Steam Reformer for Hydrogen Generation with High Efficiency and Long-Term Durability.

This paper proposes a Swiss-roll-type mini-reformer employing a copper-zinc catalyst for high-efficient SRM process. Although the commercially available copper-zinc catalysts commonly used in cylindrical-type reformers provide decent conversion rates in the short term, their long-term durability still requires improvement, mainly due to temperature variations in the reformer, catalyst loading, and thermal sintering issues. This Swiss-roll-shaped mini-reformer is designed to improve thermal energy preservation/temperature uniformity by using dual spiral channels to improve the long-term durability while maintaining methanol-reforming efficiency. It was fabricated on a copper plate that was 80 mm wide, 80 mm long, and 4 mm high with spiral channels that were 2 mm deep, 4 mm wide, and 350 mm long. To optimize the design and reformer operation, the catalyst porosity, gas hourly speed velocity (GHSV), operation temperature, and fuel feeding rate are investigated. Swiss-roll-type reformers may require higher driving pressures but can provide better thermal energy preservation and temperature uniformity, posing a higher conversion rate for the same amount of catalyst when compared with other geometries. By carefully adjusting the catalyst bed porosity, locations, and catalyst loading amount as well as other conditions, an optimized gas hourly space velocity (GHSV) can be obtained (14,580 mL/g·h) and lead to not only a high conversion rate (96%) and low carbon monoxide generation rate (0.98%) but also a better long-term durability (decay from 96% to 88.12% after 60 h operation time) for SRM processes. The decay rate, 0.13%/h, after 60 h of operation, is five-folds lower than that (0.67%/h, 0.134%/h) of a commercial cylindrical-type fixed-bed reactor with a commercial catalyst.

HeapMS: An Automatic Peak-Picking Pipeline for Targeted Proteomic Data Powered by 2D Heatmap Transformation and Convolutional Neural Networks.

The process of peak picking and quality assessment for multiple reaction monitoring (MRM) data demands significant human effort, especially for signals with low abundance and high interference. Although multiple peak-picking software packages are available, they often fail to detect peaks with low quality and do not report cases with low confidence. Furthermore, visual examination of all chromatograms is still necessary to identify uncertain or erroneous cases. This study introduces HeapMS, a web service that uses artificial intelligence to assist with peak picking and the quality assessment of MRM chromatograms. HeapMS applies a rule-based filter to remove chromatograms with low interference and high-confidence peak boundaries detected by Skyline. Additionally, it transforms two histograms (representing light and heavy peptides) into a single encoded heatmap and performs a two-step evaluation (quality detection and peak picking) using image convolutional neural networks. HeapMS offers three categories of peak picking: uncertain peak picking that requires manual inspection, deletion peak picking that requires removal or manual re-examination, and automatic peak picking. HeapMS acquires the chromatogram and peak-picking boundaries directly from Skyline output. The output results are imported back into Skyline for further manual inspection, facilitating integration with Skyline. HeapMS offers the benefit of detecting chromatograms that should be deleted or require human inspection. Based on defined categories, it can significantly reduce human workload and provide consistent results. Furthermore, by using heatmaps instead of histograms, HeapMS can adapt to future updates in image recognition models. The HeapMS is available at: https://github.com/ccllabe/HeapMS.

Cross-Binding of Adenosine by Aptamers Selected Using Theophylline.

We recently reported that some adenosine binding aptamers can also bind caffeine and theophylline with around 20-fold lower affinities. This discovery led to the current work to examine the cross-binding of adenosine to theophylline aptamers. For the DNA aptamer for theophylline, cross-binding to adenosine was observed, and the affinity was 18 to 38-fold lower for adenosine based on assays using isothermal titration calorimetry and ThT fluorescence spectroscopy. The binding complexes were characterized using NMR spectroscopy, and both adenosine and theophylline showed an overall similar binding structure to the DNA theophylline aptamer, although small differences were also observed. In contrast, the RNA aptamer did not show binding to adenosine, although both aptamers have very similar relative selectivity for various methylxanthines including caffeine. After a negative selection, a few new aptamers with completely different primary sequences for theophylline were obtained and they did not show binding to adenosine. Thus, there are many ways for aptamers to bind theophylline and some can have cross-binding to adenosine. In biology, theophylline, caffeine, and adenosine can bind to the same protein receptors to regulate sleep, and their binding to the same DNA motifs may suggest an early role of nucleic acids in similar regulatory functions.

Intestinal microbiota analysis of different Blastocystis subtypes and Blastocystis-negative individuals in Taiwan.

BACKGROUND: Intestinal parasitic infections are the most common infectious diseases among Southeast Asian migrant workers in Taiwan, especially for infections with Blastocystis hominis. However, little is known about the impact of Blastocystis subtypes (STs) on the gut microbiota. MATERIAL AND METHODS: We retrospectively evaluated the prevalence of intestinal parasites in a teaching hospital in Northern Taiwan in the period of 2015 to 2019. Blastocystis-positive stool specimens were collected for ST analysis by polymerase chain reaction in 2020. Intestinal microbiota analyses of different Blastocystis STs and Blastocystis-free individuals were conducted by 16S rRNA sequencing. RESULTS: A total of 13,859 subjects were analyzed, of which 1,802 cases (13%) were diagnosed with intestinal parasitic infections. B. hominis infections were the most prevalent (n = 1546, 85.7%). ST analysis of Blastocystis-positive samples (n=150) indicated that ST1 was the most common type, followed by ST3, ST4, ST2, ST7, and ST5. Different Blastocystis STs (ST1, ST3, and ST4) were associated with distinct richness and diversity of the microbiota. Taxonomic profiles revealed that Akkermansia muciniphila was significantly enriched for all analyzed Blastocystis STs, whereas Holdemanella biformis was more abundant in the Blastocystis-free group. Additionally, Succinivibrio dextrinosolvens and Coprococcus eutactus were specifically more abundant in ST3 carriers than in non-infected individuals. CONCLUSIONS: This study demonstrates that A. muciniphila is positively associated with all Blastocystis STs, while H. biformis was negatively associated with them. Several bacteria were enriched in specific STs, highlighting the need for further microbiota analysis at the ST level to elucidate the pathogenicity of Blastocystis.

Valorization of Glucose-Derived Humin as a Low-Cost, Green, Reusable Adsorbent for Dye Removal, and Modeling the Process.

Glucose can be isomerized into fructose and dehydrated into key platform biochemicals, following the "bio-refinery concept". However, this process generates black and intractable substances called humin, which possess a polymeric furanic-type structure. In this study, glucose-derived humin (GDH) was obtained by reacting D-glucose with an allylamine catalyst in a deep eutectic solvent medium, followed by a carbonization step. GDH was used as a low-cost, green, and reusable adsorbent for removing cationic methylene blue (MB) dye from water. The morphology of carbonized GDH differs from pristine GDH. The removal efficiencies of MB dye using pristine GDH and carbonized GDH were 52% and 97%, respectively. Temperature measurements indicated an exothermic process following pseudo-first-order kinetics, with adsorption behavior described by the Langmuir isotherm. The optimum parameters were predicted using the response surface methodology and found to be a reaction time of 600 min, an initial dye concentration of 50 ppm, and a GDH weight of 0.11 g with 98.7% desirability. The MB dye removal rate optimized through this model was 96.85%, which was in good agreement with the experimentally obtained value (92.49%). After 10 cycles, the MB removal rate remained above 80%, showcasing the potential for GDH reuse and cost-effective wastewater treatment.

Metabolomics analysis reveals changes related to pseudocyst formation induced by iron depletion in Trichomonas vaginalis.

BACKGROUND: Iron is an essential element for cellular functions, such as energy metabolism. Trichomonas vaginalis, a human urogenital tract pathogen, is capable of surviving in the environment without sufficient iron supplementation. Pseudocysts (cyst-like structures) are an environmentally tolerated stage of this parasite while encountering undesired conditions, including iron deficiency. We previously demonstrated that iron deficiency induces more active glycolysis but a drastic downregulation of hydrogenosomal energy metabolic enzymes. Therefore, the metabolic direction of the end product of glycolysis is still controversial. METHODS: In the present work, we conducted an LC‒MS-based metabolomics analysis to obtain accurate insights into the enzymatic events of T. vaginalis under iron-depleted (ID) conditions. RESULTS: First, we showed the possible digestion of glycogen, cellulose polymerization, and accumulation of raffinose family oligosaccharides (RFOs). Second, a medium-chain fatty acid (MCFA), capric acid, was elevated, whereas most detected C18 fatty acids were reduced significantly. Third, amino acids were mostly reduced, especially alanine, glutamate, and serine. Thirty-three dipeptides showed significant accumulation in ID cells, which was probably associated with the decrease in amino acids. Our results indicated that glycogen was metabolized as the carbon source, and the structural component cellulose was synthesized at same time. The decrease in C18 fatty acids implied possible incorporation in the membranous compartment for pseudocyst formation. The decrease in amino acids accompanied by an increase in dipeptides implied incomplete proteolysis. These enzymatic reactions (alanine dehydrogenase, glutamate dehydrogenase, and threonine dehydratase) were likely involved in ammonia release. CONCLUSION: These findings highlighted the possible glycogen utilization, cellulose biosynthesis, and fatty acid incorporation in pseudocyst formation as well as NO precursor ammonia production induced by iron-depleted stress.

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines.

The classical DNA aptamer for adenosine and ATP was selected twice using ATP as the target in 1995 and 2005, respectively. In 2022, this motif appeared four more times from selections using adenosine, ATP, theophylline, and caffeine as targets, suggesting that this aptamer can also bind methylxanthines. In this work, using thioflavin T fluorescence spectroscopy, this classical DNA aptamer showed Kd values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 µM, respectively, and similar Kd values were obtained using isothermal titration calorimetry. Binding to the methylxanthines was also observed for the newly selected Ade1301 aptamer but not for the Ade1304 aptamer. The RNA aptamer for ATP also had no binding to the methylxanthines. Molecular dynamics simulations were performed using the classical DNA and RNA aptamers based on their NMR structures, and the simulation results were consistent with the experimental observations, explaining the selectivity profiles. This study suggests that a broader range of target analogues need to be tested for aptamers. For the detection of adenosine and ATP, the Ade1304 aptamer is a better choice due to its better selectivity.

Surface-enhanced Raman scattering (SERS) by gold nanoparticle characterizes dermal thickening by collagen in bleomycin-treated skin ex vivo.

PURPOSE: Current skin imaging modalities, including optical, electron, and confocal microscopy, mostly require tissue fixations that could damage proteins and biological molecules. Live tissue or cell imaging such as ultrasonography and optical coherent microscope may not adequately measure the dynamic spectroscopical changes. Raman spectroscopy has been adopted for skin imaging in vivo, mostly for skin cancer imaging. However, whether the epidermal and dermal thickening in skin could be measured and distinguished by conventional Ramen spectroscopy or the surface-enhanced Raman scattering (SERS), a rapid and label-free method for noninvasive measurement remains unknown. METHODS: Human skin sections from patients of atopic dermatitis and keloid, which represent epidermal and dermal thickening, respectively, were measured by conventional Ramen spectroscopy. In mice, skin sections from imiquimod (IMQ)- and bleomycin (BLE)-treated mice, which reflect the epidermal and dermal thickening, respectively, were measured by SERS, that incorporates gold nanoparticles to generate surface plasma and enhance Raman signals. RESULTS: Conventional Ramen spectroscopy failed to consistently show the Raman shift in human samples among the different groups. SERS successfully revealed a prominent peak around 1300 cm-1 in the IMQ-treated skin; and two significant peaks around 1100 and 1300 cm-1 in BLE-treated group. Further quantitative analysis showed 1100 cm-1 peak was significantly accentuated in the BLE-treated skin than that in control skin. SERS identified in vitro a similar 1100 cm-1 peak in solutions of collagen, the major dermal biological molecules. CONCLUSION: SERS distinguishes the epidermal or dermal thickening in mouse skin with rapid and label-free measures. A prominent 1100 cm-1 SERS peak in the BLE-treated skin may result from collagen. SERS might help precision diagnosis in the future.

Convolutional Neural Network for Individual Identification Using Phase Space Reconstruction of Electrocardiogram.

Electrocardiogram (ECG) biometric provides an authentication to identify an individual on the basis of specific cardiac potential measured from a living body. Convolutional neural networks (CNN) outperform traditional ECG biometrics because convolutions can produce discernible features from ECG through machine learning. Phase space reconstruction (PSR), using a time delay technique, is one of the transformations from ECG to a feature map, without the need of exact R-peak alignment. However, the effects of time delay and grid partition on identification performance have not been investigated. In this study, we developed a PSR-based CNN for ECG biometric authentication and examined the aforementioned effects. Based on a population of 115 subjects selected from the PTB Diagnostic ECG Database, a higher identification accuracy was achieved when the time delay was set from 20 to 28 ms, since it produced a well phase-space expansion of P, QRS, and T waves. A higher accuracy was also achieved when a high-density grid partition was used, since it produced a fine-detail phase-space trajectory. The use of a scaled-down network for PSR over a low-density grid with 32 × 32 partitions achieved a comparable accuracy with using a large-scale network for PSR over 256 × 256 partitions, but it had the benefit of reductions in network size and training time by 10 and 5 folds, respectively.

Machine Learning Directed Aptamer Search from Conserved Primary Sequences and Secondary Structures.

Computer-aided prediction of aptamer sequences has been focused on primary sequence alignment and motif comparison. We observed that many aptamers have a conserved hairpin, yet the sequence of the hairpin can be highly variable. Taking such secondary structure information into consideration, a new algorithm combining conserved primary sequences and secondary structures is developed, which combines three scores based on sequence abundance, stability, and structure, respectively. This algorithm was used in the prediction of aptamers from the caffeine and theophylline selections. In the late rounds of the selections, when the libraries were converged, the predicted sequences matched well with the most abundant sequences. When the libraries were far from convergence and the sequences were deemed challenging for traditional analysis methods, this algorithm still predicted aptamer sequences that were experimentally verified by isothermal titration calorimetry. This algorithm paves a new way to look for patterns in aptamer selection libraries and mimics the sequence evolution process. It will help shorten the aptamer selection time and promote the biosensor and chemical biology applications of aptamers.

Simultaneous Detection of L-Lactate and D-Glucose Using DNA Aptamers in Human Blood Serum.

L-lactate is a key metabolite indicative of physiological states, glycolysis pathways, and various diseases such as sepsis, heart attack, lactate acidosis, and cancer. Detection of lactate has been relying on a few enzymes that need additional oxidants. In this work, DNA aptamers for L-lactate were obtained using a library-immobilization selection method and the highest affinity aptamer reached a Kd of 0.43 mM as determined using isothermal titration calorimetry. The aptamers showed up to 50-fold selectivity for L-lactate over D-lactate and had little responses to other closely related analogs such as pyruvate or 3-hydroxybutyrate. A fluorescent biosensor based on the strand displacement method showed a limit of detection of 0.55 mM L-lactate, and the sensor worked in 90 % serum. Simultaneous detection of L-lactate and D-glucose in the same solution was achieved. This work has broadened the scope of aptamers to simple metabolites and provided a useful probe for continuous and multiplexed monitoring.

Gold Nanoparticles Synthesized Using Various Reducing Agents and the Effect of Aging for DNA Sensing.

Gold nanoparticles (AuNPs) are one of the most commonly used reagents in colloidal science and biosensor technology. In this work, we first compared AuNPs prepared using four different reducing agents including citrate, glucose, ascorbate, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). At the same absorbance at the surface plasmon peak of 520-530 nm, citrate-AuNPs and glucose-AuNPs adsorbed more DNA and achieved higher affinity to the adsorbed DNA. In addition, citrate-AuNPs had better sensitivity than glucose-AuNPs for label-free DNA detection. Then, using citrate-AuNPs, the effect of aging was studied by incubation of the AuNPs at 22 °C (room temperature) and at 4 °C for up to 6 months. During aging, the colloidal stability and DNA adsorption efficiency gradually decreased. In addition, the DNA sensing sensitivity using a label-free method also dropped around 4-fold after 6 months. Heating at boiling temperature of the aged citrate-AuNPs could not rejuvenate the sensing performance. This study shows that while citrate-AuNPs are initially better than the other three AuNPs in their colloid properties and sensing properties, this edge in performance might gradually decrease due to constantly changing surface properties caused from the aging effect.

Chiral weak ferromagnets formed in one-dimensional organic-inorganic hybrid manganese chloride hydrates.

Chiral weak ferromagnets were developed in organic-inorganic hybrid systems of one-dimensional manganese(II) chloride hydrate with chiral organic cations of (S)- and (R)-ß-methylphenethylammonium (= (S)- and (R)-MPA+), [(S)/(R)-MPA]2[MnCl4(H2O)]. The weak ferromagnetic phase found below 3.2 K could be attributed to a spin canting antiferromagnetic state induced by Dzyaloshinskii-Moriya interactions owing to spatial inversion symmetry breaking in chirality introduced systems. This is the first chiral weak ferromagnet found in a spin chain of deformed perovskite derivatives with a corner-sharing structure of transition metal halide hydrates.

Protection of DNA by metal ions at 95 °C: from lower critical solution temperature (LCST) behavior to coordination-driven self-assembly.

While polyvalent metal ions and heating can both degrade nucleic acids, we herein report that a combination of them leads to stabilization. After incubating 4 mM various metal ions and DNA oligonucleotides at 95 °C for 3 h at pH 6 or 8, metal ions were divided into four groups based on gel electrophoresis results. Mg2+ can stabilize DNA at pH 6 without forming stable nanoparticles at room temperature. Co2+, Cu2+, Cd2+, Mn2+ and Zn2+ all protected the DNA and formed nanoparticles, whereas the nanoparticles formed with Fe2+ and Ni2+ were so stable that they remained even in the presence of EDTA. At pH 8, Ce3+ and Pb2+ showed degraded DNA bands. For Mg2+, better protection was achieved with higher metal and DNA concentrations. By monitoring temperature-programmed fluorescence change, a sudden drop in fluorescence intensity attributable to the lower critical solution temperature (LCST) transition of DNA was found to be around 80 °C for Mg2+, while this transition temperature decreased with increasing Mn2+ concentration. The unexpected thermal stability of DNA enabled by metal ions is useful for extending the application of DNA at high temperatures, forming coordination-driven nanomaterials, and it might offer insights into the origin of life on the early Earth.

A DNA Aptamer for Theophylline with Ultrahigh Selectivity Reminiscent of the Classic RNA Aptamer.

Since the report of the RNA aptamer for theophylline, theophylline has become a key molecule in chemical biology for designing RNA switches and riboswitches. In addition, theophylline is an important drug for treating airway diseases including asthma. The classic RNA aptamer with excellent selectivity for theophylline has been used to design biosensors, although DNA aptamers are more desirable for stability and cost considerations. In this work, we selected DNA aptamers for theophylline, and all the top sequences shared the same binding motifs. Binding was confirmed using isothermal titration calorimetry and a nuclease digestion assay, showing a dissociation constant (Kd) around 0.5 µM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a Kd of 9.8 µM. The selectivity for theophylline over caffeine is around 250,000-fold based on a strand-displacement assay, which was more than 20-fold higher compared to the classic RNA aptamer. For other tested analogs, the DNA aptamer also showed better selectivity. Using the structure-switching aptamer sensor design method, a detection limit of 17 nM theophylline was achieved in the selection buffer, and a detection limit of 31 nM was obtained in 10% serum.

Label-free and Dye-free Fluorescent Sensing of Tetracyclines Using a Capture-Selected DNA Aptamer.

Tetracyclines are a group of important antibiotics with a common four-ring scaffold. While most tetracyclines are currently used only in animals, their leaching into the environment and residues in food have caused health concerns. Aptamers are an attractive way to detect tetracyclines, and all previously reported aptamers for tetracyclines were obtained by immobilizing target molecules. In this work, we selected a few DNA aptamers by immobilizing the DNA library using oxytetracycline as the target. We obtained new aptamers with no overlapping sequences compared to the previously reported ones, and a representative sequence named OTC5 had a dissociation constant of 147 nM measured by isothermal titration calorimetry. Similar binding affinities were also observed with tetracycline and doxycycline. Because tetracyclines are fluorescent and their fluorescence intensity was enhanced by binding to the aptamers, a label-free and dye-free fluorescent biosensor was developed with a detection limit of 25 nM oxytetracycline. The sensor was able to detect targets in milk after extraction. Fluorescence polarization measurement showed that this aptamer is insensitive to sodium concentration but requires magnesium. Finally, a strand-displacement biosensor was designed, and it has a detection limit of 1.2 µM oxytetracycline.

Comprehensive functional genomic analyses link APC somatic mutation and mRNA-miRNA networks to the clinical outcome of stage-III colorectal cancer patients.

BACKGROUND: Colorectal cancer (CRC) is a major health concern globally, but exhibits regional and/or environmental distinctions in terms of outcome especially for patients with stage III CRC. METHODS: From 2014 to 2016, matched pairs of tumor and adjacent normal tissue samples from 60 patients with stage I-IV CRC from Chang Gung Memorial Hospital in Taiwan were analyzed using next-generation sequencing. The DNA, mRNA, and miRNA sequences of paired tumor tissues were profiled. An observational study with survival analysis was done. Online datasets of The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC) were also integrated and compared. RESULTS: The gene that exhibited the highest mutation rate was adenomatous polyposis coli (APC) (75.0%), followed by TP53 (70.0%), KRAS (56.6%), and TTN (48.3%). APC was also the most frequently mutated gene in TCGA and ICGC datasets. Surprisingly, for non-metastatic cases (stages I-III), CRC patients with mutated APC had better outcome in terms of overall survival (p = 0.041) and recurrence free survival (p = 0.0048). Particularly for stage III CRC, the overall survival rate was 94.4% and 67.7%, respectively (p = 0.018), and the recurrence free survival rate was 94.4% and 16.7%, respectively (p = 0.00044). Further clinical and gene expression analyses revealed that the APC wt specimens to a greater extent exhibit poor differentiation state as well as EGFR upregulation, providing molecular basis for the poor prognosis of these patients. Finally, based on integrated transcriptome analysis, we constructed the mRNA-miRNA networks underlying disease recurrence of the stage III CRC and uncovered potential therapeutic targets for this clinical condition. CONCLUSION: For stage III CRC, patients with mutated APC had better overall and recurrence free survival.

2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer.

Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a Kd of 15.0 mM glucose, which was comparable with the 10 mM Kd previously reported using the strand displacement assay. The binding required two Na+ ions or one Mg2+ that cannot be replaced by Li+ or K+ . The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher Kd values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.