Development of fecal microbial diagnostic marker sets of colorectal cancer using natural language processing method.

BACKGROUND: Cancer screening and early detection greatly increase the chances of successful treatment. However, most cancer types lack effective early screening biomarkers. In recent years, natural language processing (NLP)-based text-mining methods have proven effective in searching the scientific literature and identifying promising associations between potential biomarkers and disease, but unfortunately few are widely used. METHODS: In this study, we used an NLP-enabled text-mining system, MarkerGenie, to identify potential stool bacterial markers for early detection and screening of colorectal cancer. After filtering markers based on text-mining results, we validated bacterial markers using multiplex digital droplet polymerase chain reaction (ddPCR). Classifiers were built based on ddPCR results, and sensitivity, specificity, and area under the curve (AUC) were used to evaluate the performance. RESULTS: A total of 7 of the 14 bacterial markers showed significantly increased abundance in the stools of colorectal cancer patients. A five-bacteria classifier for colorectal cancer diagnosis was built, and achieved an AUC of 0.852, with a sensitivity of 0.692 and specificity of 0.935. When combined with the fecal immunochemical test (FIT), our classifier achieved an AUC of 0.959 and increased the sensitivity of FIT (0.929 vs. 0.872) at a specificity of 0.900. CONCLUSIONS: Our study provides a valuable case example of the use of NLP-based marker mining for biomarker identification.

Postoperative circulating tumor DNA detection and CBLB mutations are prognostic biomarkers for gastric cancer.

BACKGROUND: Several studies have demonstrated that circulating tumor DNA (ctDNA) can be used to predict the postoperative recurrence of several cancers. However, there are few studies on the use of ctDNA as a prognosis tool for gastric cancer (GC) patients. OBJECTIVE: This study aims to determine whether ctDNA could be used as a prognostic biomarker in GC patients through multigene-panel sequencing. METHODS: Using next-generation sequencing (NGS) Multigene Panels, the mutational signatures associated with the prognosis of GC patients were identified. We calculated the survival probability with Kaplan-Meier and used the Log-rank test to compare survival curves between ctDNA-positive and ctDNA-negative groups. Potential application of radiology combined with tumor plasma biomarker analysis of ctDNA in GC patients was carried out. RESULTS: Disease progression is more likely in ctDNA-positive patients as characterized clinically by a generally higher T stage and a poorer therapeutic response (P < 0.05). ctDNA-positive patients also had worse overall-survival (OS: P = 0.203) and progression-free survival (PFS: P = 0.037). The combined analysis of ctDNA, radiological, and serum biomarkers in four patients indicated that ctDNA monitoring can be a good complement to radiological and plasma tumor markers for GC patients. Kaplan-Meier analysis using a cohort of GC patients in the TCGA database showed that patients with CBLB mutations had shorter OS and PFS than wild-type patients (OS: P = 0.0036; PFS: P = 0.0027). CONCLUSIONS: This study confirmed the utility and feasibility of ctDNA in the prognosis monitoring of gastric cancer.

A fluorescent sensor based on the cascade signal amplification strategy for ultra-sensitive detection of Cu2.

Copper is an essential element in the human body, participating in various physiological activities in the bodies of organisms. However, an excessive load of Cu2+ is associated with several neurodegenerative diseases and prion diseases, also identified as a symptom of Wilson's disease (WD). A straightforward, rapid, sensitive, and specific copper sensor is highly required but remains a challenge. In this study, guided by the simulation, we developed a chemical sensor using a cascade signal amplification strategy based on the Cu-catalyzed click reaction, combined with a fluorescence-enhanced substrate with gold nanorods coupled with silver nanoislands. The sensor can selectively detect Cu2+ as low as 3.87 nM within 10 min. We have demonstrated that this method can be directly employed for WD diagnosis in urine samples. In addition, using antibiotic susceptibility testing (AST) as an example, we verify whether this assay can be adapted to other targets where Cu is designed as an indirect indicator.

Rapid In Situ Hydrogel LAMP for On-Site Large-Scale Parallel Single-Cell HPV Detection.

Rapid human papillomavirus (HPV) screening is urgently needed for preventing and early diagnosis of cervical cancer in rural areas. To date, no HPV nucleic acid test (NAT) can be implemented within a single patient visit starting from clinical samples. Here, we develop a hydrogel loop-mediated isothermal amplification (LAMP) method in a fashion of large-scale parallel (about 1000 cells) in situ HPV DNA detection in clinical cervical exfoliated cells at the single-cell level. It can be used with a hotplate and smartphone to obtain HPV NAT results in less than 30 min, which is especially suitable for the on-site scenario. We apply this rapid HPV NAT on 40 clinical cervical exfoliated cell samples and compare the results to a clinical gold standard quantitative polymerase chain reaction (qPCR) method [area under curve (AUC), 1.00]. Meanwhile, our assay can provide HPV infection information for large-scale parallel single clinical cervical exfoliated cells, which cannot be received from traditional NAT methods. Our findings suggest the potential of in situ hydrogel LAMP as a powerful tool for clinical HPV screening and fundamental research.

Bioinspired Plasmonic Nanosensor for on-Site Antimicrobial Susceptibility Testing in Urine Samples.

Delayed use of appropriate antibiotics for superbugs, particularly for extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pn), has caused extensive morbidity and mortality worldwide. Therefore, rapid and on-site antimicrobial susceptibility testing (AST) is urgently required. Unfortunately, currently, no phenotypic AST can realize a sample-to-answer result within 2 h directly from a clinical sample and without using laboratory equipment or customized devices. Inspired by observing that E. coli and K. pn can rapidly catalyze H2O2, we developed a plasmonic nanosensor that responds to the proliferation of bacteria for realizing rapid AST. The results can be determined with the naked eye, digitized using a smartphone, and validated using ultraviolet-visible spectrometry. Our assay achieved superb area under the curves of 0.9752 and 1 in a receiver operating characteristic analysis directly obtained from uncultured clinical urine samples infected by E. coli and K. pn, respectively. The entire process from sample collection to analysis takes 100 min for E. coli and 85 min for K. pn detection. Our platform provides a practical approach for performing on-site AST in clinics to improve the survival of patients. It releases the burden of superbugs and avoids the abuse of antibiotics.

Dynamic Alteration of the Gut Microbiota Associated with Obesity and Intestinal Inflammation in Ovariectomy C57BL/6 Mice.

OBJECTIVE: Estrogen is a critical hormone that is mainly produced by the ovary in females. Estrogen deficiency leads to various syndromes and diseases, partly due to gut microbiota alterations. Previous studies have shown that estrogen deficiency affects the gut microbiota at 6-8 weeks after ovariectomy, but the immediate effect of estrogen deficiency on the gut microbiota remains poorly understood. METHODS: To investigate the short time and dynamic effects of decreased estrogen levels on the gut microbiota and their potential impact on estrogen deficiency-related diseases, we performed metagenomic sequencing of 260 fecal samples from 50 ovariectomy (OVX) and 15 control C57BL/6 female mice at four time points after surgery. RESULTS: We found that seven gut microbiota species, including E. coli, Parabacteroides unclassified, Lachnospiraceae bacterium 8_1_57FAA, Bacteroides uniformis, Veillonella unclassified, Bacteroides xylanisolvens, and Firmicutes bacterium M10_2, were abundant in OVX mice. The abundance of these species increased with time after OVX surgery. The relative abundance of the opportunistic pathogen E. coli and the Crohn's disease-related Veillonella spp. was significantly correlated with mouse weight gain in the OVX group. Butyrate production and the Entner-Doudoroff pathway were significantly enriched in the control mouse group, while the degradation of glutamic acid and aspartic acid was enriched in the OVX mouse group. As the time after OVX surgery increased, the bacterial species and metabolic pathways significantly changed and tended to suggest an inflammatory environment, indicating a subhealthy state of the gut microbiota in the OVX mouse group. CONCLUSIONS: Taken together, our results show that the dynamic gut microbiota profile alteration caused by estrogen deficiency is related to obesity and inflammation, which may lead to immune and metabolic disorders. This study provides new clues for the treatment of estrogen deficiency-related diseases.

Inhibition of GSK3 Represses the Expression of Retinoic Acid Synthetic Enzyme ALDH1A2 via Wnt/ß-Catenin Signaling in WiT49 Cells.

Organogenesis, including renal development, requires an appropriate retinoic acid concentration, which is established by differential expression of aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and cytochrome P450 family 26 subfamily A/B/C member 1 (CYP26A1/B1/C1). In the fetal kidney, ALDH1A2 expresses in the developing stroma and renal vesicle and its derivatives but does not present in the ureteric bud. It remains unclear what may contribute to this expression pattern. Here we show that the glycogen synthase kinase 3 alpha/beta (GSK3A/B) inhibitor CHIR99021 significantly represses ALDH1A2 expression in WiT49, which is a Wilms' tumor cell line that exhibits "triphasic" differential potential and is used as a fetal kidney cell model. CHIR99021 fails to suppress ALDH1A2 as ß-catenin is inhibited, suggesting that the downregulation of ALDH1A2 by CHIR99021 is through Wnt/ß-catenin signaling. Ectopic expression of mouse Wnt1, Wnt3a, Wnt4, and Wnt9b represses ALDH1A2 expression in WiT49 cells. Using immunohistochemistry, we show an inverse correlation of Aldh1a2 expression with ß-catenin in rat E18.5 kidney. ChIP demonstrated that ß-catenin is recruited to the ALDH1A2 promoter, the conserved intron1G, and another site within intron 1 of ALDH1A2. Using a luciferase assay, we further show that the ALDH1A2 promoter and the intron1G element are involved in the repression of ALDH1A2 expression by CHIR99021. Our work demonstrates that ALDH1A2 expression can be directly repressed by the Wnt/ß-catenin signaling in fetal kidney cells, suggesting that Wnt/ß-catenin may play a role in maintaining the expression pattern of ALDH1A2 in the fetal kidney, thus controlling the availability and localization of retinoic acid and regulating aspects of kidney development.

Breast Cancer Subtype Classification Using 4-Plex Droplet Digital PCR.

BACKGROUND: Infiltrating ductal carcinoma (IDCA) is the most common form of invasive breast cancer. Immunohistochemistry (IHC) is widely used to analyze estrogen receptor 1 (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) that can help classify the tumor to guide the medical treatment. IHC examinations require experienced pathologists to provide interpretations that are subjective, thereby lowering the reproducibility of IHC-based diagnosis. In this study, we developed a 4-plex droplet digital PCR (ddPCR) for the simultaneous and quantitative analyses of estrogen receptor 1 (ESR1), progesterone receptor (PGR), erb-b2 receptor tyrosine kinase 2 (ERBB2), and pumilio RNA binding family member 1 (PUM1) expression levels in formalin-fixed paraffin-embedded (FFPE) samples. METHODS: We evaluated the sensitivity, reproducibility, and linear dynamic range of 4-plex ddPCR. We applied this method to analyze 95 FFPE samples from patients with breast IDCA and assessed the agreement rates between ddPCR and IHC to evaluate its potential in classifying breast cancer subtypes. RESULTS: The limits of quantification (LOQ) were 25, 50, 50, and 50 copies per reaction for ERBB2, ESR1, PGR, and PUM1, respectively. The dynamic ranges of ESR1, PGR, and PUM1 extended over 50-1600 copies per reaction and those of ERBB2 from 25 to 1600 copies per reaction. The concordance correlation coefficients between 4-plex ddPCR and IHC were 96.8%, 91.5%, and 85.1% for ERBB2, ESR1, and PGR, respectively. Receiver operating characteristic curve area under the curve values of 0.991, 0.977, and 0.920 were generated for ERBB2, ESR1, and PGR, respectively. CONCLUSIONS: Evaluation of breast cancer biomarker status by 4-plex ddPCR was highly concordant with IHC in this study.

High-throughput blood sample preparation for single nucleotide polymorphism genotyping in less than 25 min.

Straightforward, rapid and high-throughput pretreatment for single nucleotide polymorphisms (SNP) genotyping is critically needed in clinical practice. However, all existing SNP genotyping methods require DNA purification step, which is labor-intensive and time-consuming. We develop a protocol for SNP genotyping by combining whole blood lysis (WBL) with qPCR and justify the practicality of our method in blood samples from 140 donors, including 40 samples from healthy donors, and 100 samples from donors with either low white blood cell counts, high level of serum uric acid or triglyceride. When compared with Sanger sequencing, the gold standard for SNP genotyping, our method exhibits a 100% consistency in the aspect of sensitivity and specificity. In addition, our method can obtain amplifiable DNA within 25 mins (which is the fastest to the best of our knowledge) from 48 samples. The blood samples, even with low white blood cell counts, high level of serum uric acid or triglyceride could not affect the sensitivity and specificity of our method. Our study demonstrates that the combination of WBL and qPCR genotyping can serve as a high-throughput and robust approach for routine clinical SNP genotyping.

miR­873 inhibits colorectal cancer cell proliferation by targeting TRAF5 and TAB1.

MicroRNA-873 (miR­873) has been reported to be dysregulated in a variety of malignancies, however, the biological function and underlying molecular mechanism of miR­873 in colorectal cancer (CRC) remain unclear. In the present study we found that the expression levels of miR­873 were markedly decreased in CRC cell lines and tissues from patients. Statistical analysis revealed that miR­873 expression was inversely correlated with the disease stage of CRC. Kaplan­Meier survival analysis revealed that patients with CRC with lower miR­873 expression had shorter overall survival rates. Additionally, downregulation of miR­873 enhanced the proliferation of CRC cells, while upregulation of miR­873 reduced this proliferation. Furthermore, we found that tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5) and TGF­ß activated kinase 1 (MAP3K7) binding protein 1 (TAB1) were direct targets of miR­873 in CRC cells. A luciferase assay revealed that ectopic expression of miR­873 significantly reduced nuclear factor κB (NF­κB) luciferase activity, while ectopic expression of miR­873 inhibitor enhanced luciferase activity, suggesting that downregulation of miR­873 can activate NF­κB signaling. Therefore, our findings established a tumor-suppressive role for miR­873 in the inhibition of CRC progression, which may be employed as a novel prognostic marker and as an effective therapeutic target for CRC.

Regulation of retinoic acid synthetic enzymes by WT1 and HDAC inhibitors in 293 cells.

All-trans retinoic acid (atRA), which is mainly generated endogenously via two steps of oxidation from vitamin A (retinol), plays an indispensible role in the development of the kidney and many other organs. Enzymes that catalyze the oxidation of retinol to generate atRA, including aldehyde dehydrogenase 1 family (ALDH1)A1, ALDH1A2 and ALDH1A3, exhibit complex expression patterns at different stages of renal development. However, molecular triggers that control these differential expression levels are poorly understood. In this study, we provide in vitro evidence to demonstrate that Wilms' tumor 1 (WT1) negatively regulates the expression of the atRA synthetic enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, in the 293 cell line, leading to significant blockage of atRA production. Furthermore, we demonstrate that the suppression of ALDH1A1 by WT1 can be markedly attenuated by histone deacetylase inhibitors (HDACis). Taken together, we provide evidence to indicate that WT1 and HDACs are strong regulators of endogenous retinoic acid synthetic enzymes in 293 cells, indicating that they may be involved in the regulation of atRA synthesis.

A Modified Carbon Monoxide Breath Test for Measuring Erythrocyte Lifespan in Small Animals.

This study was to develop a CO breath test for RBC lifespan estimation of small animals. The ribavirin induced hemolysis rabbit models were placed individually in a closed rebreath cage and air samples were collected for measurement of CO concentration. RBC lifespan was calculated from accumulated CO, blood volume, and hemoglobin concentration data. RBC lifespan was determined in the same animals with the standard biotin-labeling method. RBC lifespan data obtained by the CO breath test method for control (CON, 49.0 ± 5.9 d) rabbits, rabbits given 10 mg/kg·d(-1) of ribavirin (RIB10, 31.0 ± 4.0 d), and rabbits given 20 mg/kg·d(-1) of ribavirin (RIB20, 25.0 ± 2.9 d) were statistically similar (all p > 0.05) to and linearly correlated (r = 0.96, p < 0.01) with the RBC lifespan data obtained for the same rabbits by the standard biotin-labeling method (CON, 51.0 ± 2.7 d; RIB10, 33.0 ± 1.3 d; and RIB20, 27.0 ± 0.8 d). The CO breath test method takes less than 3 h to complete, whereas the standard method requires at least several weeks. In conclusion, the CO breath test method provides a simple and rapid means of estimating RBC lifespan and is feasible for use with small animal models.

Cordycepin induces apoptosis and autophagy in human neuroblastoma SK-N-SH and BE(2)-M17 cells.

Cordycepin, also termed 3'-deoxyadenosine, is a derivative of the nucleoside adenosine that represents a potential novel class of anticancer drugs targeting the 3' untranslated region of RNAs. Cordycepin has been reported to induce apoptosis in certain cancer cell lines, but the effects of cordycepin on human neuroblastoma cells have not been studied. In the present study, an MTT assay revealed that cordycepin inhibits the viability of neuroblastoma SK-N-SH and BE(2)-M17 cells in a dose-dependent manner. In addition, cordycepin increases the early-apoptotic cell population of SK-N-SH cells, as determined by fluorescence-activated cell sorting analysis. The induction of apoptosis in neuroblastoma cells by cordycepin was further confirmed by western blotting, which revealed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase 1 in the SK-N-SH and BE(2)-M17 cells. Cordycepin also induced the formation of a punctate pattern of light-chain 3 (LC3)-associated green fluorescence in the SK-N-SH cells transfected with a pEGFP-LC3 vector. Furthermore, western blotting revealed cleavage of LC3 A/B in cordycepin-treated neuroblastoma SK-N-SH cells. Taken together, the results indicate that cordycepin significantly increases apoptosis and autophagy in neuroblastoma cells, and may therefore be a drug candidate for neuroblastoma therapy, but requires additional evaluation.

Epidermal overexpression of transgenic ΔNp63 promotes type 2 immune and myeloid inflammatory responses and hyperplasia via NF-κB activation.

ΔNp63 is known to be critical in skin development and cancer; however, how it triggers proliferation and inflammation in vivo remains to be elucidated. Here, we find that induced ΔNp63 expression in skin of transgenic mice (TG) results in a hyperproliferative epidermis coupled with inflammatory infiltrates. In situ, infiltrating cells include CD45(+) leukocytes, CD19(+) B lymphocytes, CD3(+) T lymphocytes, CD4(+) T helper, CD25(+)/Foxp3(+) Treg, Ly6B(+) neutrophils, S-100(+) dendritic cells, and macrophages bearing CD11b(+), F4/80(+), CD68(+), and CD206(+) M2 type markers. Transcriptional profiling of TG skin revealed increased gene expression involved in inflammation and immune responses, including Th2/M2 cytokines and chemokines. These genes were co-regulated by ΔNp63 and NF-κB RelA or cRel, and enhanced by TNF-α. Elevated cRel, RelA, and IKKs were observed in TG mouse skin and human squamous carcinomas with ΔNp63 overexpression. Thus, our findings unveil a missing link connecting overexpressed ΔNp63 with aberrant NF-κB activation, pro-inflammatory and type 2 cytokines and chemokines, and host infiltrates during skin inflammation and hyperplasia. Our findings provide a missing link between ΔNp63 overexpression and NF-κB-mediated inflammation, of potential relevance to the pathogenesis of squamous carcinoma.

Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo.

Pancreatic cancer is highly resistant to the currently available chemotherapeutic agents. Less than 5% of patients diagnosed with this disease could survive beyond 5 years. Thus, there is an urgent need for the development of novel, efficacious drugs that can treat pancreatic cancer. Herein we report the identification of artesunate (ART), a derivative of artemisinin, as a potent and selective antitumor agent against human pancreatic cancer cells in vitro and in vivo. ART exhibits selective cytotoxic activity against Panc-1, BxPC-3 and CFPAC-1 pancreatic cancer cells with IC(50) values that are 2.3- to 24-fold less than that of the normal human hepatic cells (HL-7702). The pan caspase inhibitor zVAD-fmk did not inhibit the cytotoxic activity of ART. Electron microscopy of ART-treated cells revealed severe cytoplasmic swelling and vacuolization, swollen and internally disorganized mitochondria, dilation (but not fragmentation) of the nuclei without chromatin condensation, and cell lysis, yielding a morphotype that is typical of oncosis. The ART-treated cells exhibited a loss of mitochondrial membrane potential (DeltaPsim) and ART-induced cell death was inhibited in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC). Importantly, ART produced a dose-dependent tumor regression in an in vivo pancreatic cancer xenografts model. The in vivo antitumor activity of ART was similar to that of gemcitabine. Taken together, our study suggests that ART exhibits antitumor activity against human pancreatic cancer via a novel form of oncosis-like cell death, and that ART should be considered a potential therapeutic candidate for treating pancreatic cancer.