CRISPR/Cas12b assisted loop-mediated isothermal amplification for easy, rapid and sensitive quantification of chronic HBV DNA in one-pot.

BACKGROUND: Currently, millions of people suffer from undiagnosed chronic hepatitis B (CHB) infection each year, which leads to high mortality rates attributed to cirrhosis and hepatocellular carcinoma. Previously reported assays, such as PCR-based assays, have limitations in terms of convenient for CHB screening in high-burden regions and resource-limited settings. Recently, diagnosis based on CRISPR/Cas, which has been considered as a potential method of point-of-care test (POCT) in resource-limited settings, offers a significant advantage in terms of high sensitivity and specificity. Therefore, there is an urgent need for the hepatitis B virus (HBV) detection utilizing CRISPR/Cas system. RESULTS: We have proposed a one-pot of one-step method for CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) to facilitate the quick, sensitive, and precise quantification of HBV DNA. This method is designed for point-of-care testing following genomic extraction or sample heat treatment. We have optimized several critical factors, such as the reaction buffer, AapCas12b-gRNA concentration, reporter and its concentration, reaction temperature, and chemical additives, to significantly enhance the performance of the one-pot assay for HBV. Importantly, it exhibited no cross-reactivity between HBV and blood-borne pathogens. Moreover, the assay is capable of quantifying HBV DNA within 1 h with a limit of detection (LOD) of 25 copies per milliliter. Additionally, when tested on 236 clinical samples, the assay demonstrated a sensitivity of 99.00 % (198/200) and a specificity of 100.00 % (36/36) at the 99 % confidence level compared to real-time quantitative PCR. SIGNIFICANCE: The utilization of convenient and reliable point-of-care diagnostic methods is crucial for reducing the burden of CHB globally. The assay we developed was helpful to improve the ability of HBV diagnosis for practical clinical translation, especially in high-burden regions and resource-limited settings. It has great advantages for rapid screening of CHB as well as evaluation of therapeutic efficacy as a companion diagnostic method.

The impact of sarcopenia on overall survival in patients with pan-RAS wild-type colorectal liver metastasis receiving hepatectomy.

Sarcopenia has been associated with conventional chemotherapy-related toxicity, postoperative complications and poor overall survival in patients with genotype-unselected metastatic colorectal cancer (mCRC). This study aimed to evaluate the prognostic implications of sarcopenia and its change after perioperative cetuximab plus doublet chemotherapy and hepatectomy in patients with RAS wild-type colorectal liver metastasis (CRLM). Patients with CRLM from 2007 to 2018 in Chang Gung Research Database were retrospectively analyzed. Baseline characteristics as well as skeletal muscle index (SMI) at baseline and dynamic changes after interventions were collected. A multivariate Cox proportional hazard model was used to evaluate the effect of each parameter on overall survival (OS), and the Kaplan-Meier method was used to establish survival curves. A two-sided p value < 0.05 was considered statistically significance. Of 214 RAS wild-type mCRC patients who received both cetuximab and doublet chemotherapy, 77 who received upfront or subsequent hepatectomy were included in this study. The median follow-up time was 2.3 years. The rate of sarcopenia was higher in the patients who received neoadjuvant cetuximab-containing regimens than in those who received upfront hepatectomy (95% versus 63%, p = 0.001). Increased SMI after perioperative systemic therapy remained independently associated with better OS in multivariate analysis [hazard ratio (HR) = 0.27/10% increase, p = 0.013). The patients with sarcopenia had a trend of worse OS than those without sarcopenia (median OS: 4.5 versus 3.6 years, log-rank p = 0.282). Improvement in sarcopenia ([SMI after intervention - initial SMI]/initial SMI × 100%) is an important prognostic factor for OS. Future research is warranted to investigate direct interventions for sarcopenia and the impact on OS.

Cross Platform Analysis of Volatile Organic Compounds Using Selected Ion Flow Tube and Proton-Transfer-Reaction Mass Spectrometry.

Volatile breath metabolites serve as potential disease biomarkers. Online mass spectrometry (MS) presents real-time quantification of breath volatile organic compounds (VOCs). The study aims to assess the relationship between two online analytical mass spectrometry techniques in the quantification of target breath metabolites: selected ion flow tube mass spectrometry (SIFT-MS) and proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS). The two following techniques were employed: (i) direct injection with bag sampling using SIFT-MS and PTR-ToF-MS and (ii) direct injection and thermal desorption (TD) tube comparison using PTR-ToF-MS. The concentration of abundant breath metabolites, acetone and isoprene, demonstrated a strong positive linear correlation between both mass spectrometry techniques (r = 0.97, r = 0.89, respectively; p < 0.001) and between direct injection and TD tube (r = 0.97, r = 0.92, respectively; p < 0.001) breath sampling techniques. This was reflected for the majority of short chain fatty acids and alcohols tested (r > 0.80, p < 0.001). Analyte concentrations were notably higher with the direct injection of a sampling bag compared to the TD method. All metabolites produced a high degree of agreement in the detection range of VOCs between SIFT-MS and PTR-ToF-MS, with the majority of compounds falling within 95% of the limits of agreement with Bland-Altman analysis. The cross platform analysis of exhaled breath demonstrates strong positive correlation coefficients, linear regression, and agreement in target metabolite detection rates between both breath sampling techniques. The study demonstrates the transferability of using data outputs between SIFT-MS and PTR-ToF-MS. It supports the implementation of a TD platform in multi-site studies for breath biomarker research in order to facilitate sample transport between clinics and the laboratory.

Mass-Spectrometry Analysis of Mixed-Breath, Isolated-Bronchial-Breath, and Gastric-Endoluminal-Air Volatile Fatty Acids in Esophagogastric Cancer.

A noninvasive breath test has the potential to improve survival from esophagogastric cancer by facilitating earlier detection. This study aimed to investigate the production of target volatile fatty acids (VFAs) in esophagogastric cancer through analysis of the ex vivo headspace above underivatized tissues and in vivo analysis within defined anatomical compartments, including analysis of mixed breath, isolated bronchial breath, and gastric-endoluminal air. VFAs were measured by PTR-ToF-MS and GC-MS. Levels of VFAs (acetic, butyric, pentanoic, and hexanoic acids) and acetone were elevated in ex vivo experiments in the headspace above esophagogastric cancer compared with the levels in samples from control subjects with morphologically normal and benign conditions of the upper gastrointestinal tract. In 25 patients with esophagogastric cancer and 20 control subjects, receiver-operating-characteristic analysis for the cancer-specific VFAs butyric acid ( P < 0.001) and pentatonic acid ( P = 0.005) within in vivo gastric-endoluminal air gave an area under the curve of 0.80 (95% confidence interval of 0.65 to 0.93, P = 0.01). Compared with mixed- and bronchial-breath samples, all examined VFAs were found in highest concentrations within esophagogastric-endoluminal air. In addition, VFAs were higher in all samples derived from cancer patients compared with in the controls. Equivalence of VFA levels within the mixed and bronchial breath of cancer patients suggests that their origin within breath is principally derived from the lungs and, by inference, from the systemic circulation as opposed to direct passage from the upper gastrointestinal tract. These findings highlight the potential to utilize VFAs for endoluminal-gas biopsies and noninvasive mixed-exhaled-breath testing for esophagogastric-cancer detection.

Status for clinically complete remission rectal cancer after concomitant chemo-radiotherapy in Taiwan.

Treatment for Rectal cancer changed after the induction of concomitant chemo-radiotherapy, CCRT. Complete remission of the tumor leads to debate of the necessity of surgical intervention. We evaluate the treatment outcome to know if operation is beneficial to these patients. Patients received long course concomitant chemo-radiotherapy for advanced rectal cancer between 2004 and 2013 in Taiwan were enrolled. Total 2780 patients diagnosed advanced rectal cancer were enrolled. In these patients, 2578 received surgical intervention and 202 were in wait and see for complete remission tumor. Higher local recurrence rate was found with wait and see group (8.9% vs. 2.7%). Also, better overall survival, disease free survival and local recurrence free survival were seen with the surgical intervention group. Surgical intervention may be benefit for some misdiagnosed completed response to CCRT.

Circulating Tumor Cell Count Correlates with Colorectal Neoplasm Progression and Is a Prognostic Marker for Distant Metastasis in Non-Metastatic Patients.

Enumeration of circulating tumor cells (CTCs) has been proven as a prognostic marker for metastatic colorectal cancer (m-CRC) patients. However, the currently available techniques for capturing and enumerating CTCs lack of required sensitivity to be applicable as a prognostic marker for non-metastatic patients as CTCs are even more rare. We have developed a microfluidic device utilizing antibody-conjugated non-fouling coating to eliminate nonspecific binding and to promote the multivalent binding of target cells. We then established the correlation of CTC counts and neoplasm progression through applying this platform to capture and enumerate CTCs in 2 mL of peripheral blood from healthy (n = 27), benign (n = 21), non-metastatic (n = 95), and m-CRC (n = 15) patients. The results showed that the CTC counts progressed from 0, 1, 5, to 36. Importantly, after 2-year follow-up on the non-metastatic CRC patients, we found that those who had ≥5 CTCs were 8 times more likely to develop distant metastasis within one year after curable surgery than those who had <5. In conclusion, by employing a sensitive device, CTC counts show good correlation with colorectal neoplasm, thus CTC may be as a simple, independent prognostic marker for the non-metastatic CRC patients who are at high risk of early recurrence.

MicroRNA-223 and microRNA-92a in stool and plasma samples act as complementary biomarkers to increase colorectal cancer detection.

Aberrant levels of circulating miRNAs are potential biomarkers for the early detection of colorectal cancer (CRC). However, no previous systematic study has examined miRNAs in various specimen types from the same patient to evaluate their clinical utility. In this study, we compiled information from ~450 articles published before 2012, and selected the 46 most frequently reported CRC-related miRNAs as candidates. We then established a 46-miRNA multiplex RT-qPCR method, and efficiently examined two clinically accessible samples: stool from fecal occult blood test and EDTA plasma. A total of 62 tissue, 447 stool, and 398 plasma samples were collected from CRC patients and healthy controls. Good correlations of detectable miRNAs were noticed in paired tumor tissues, stool, and plasma samples of 62 CRC patients. Using these 62 CRC patients and 62 matched healthy controls as the training set, 5 and 11 differentially expressed miRNAs achieved the area under the ROC curve (AUC) greater than 0.7 in stool and plasma samples, respectively. The selected miRNAs was subsequently validated using the remaining enrolled samples as the test cohort; 4 miRNAs in stool and 6 miRNAs in plasma were maintained discriminating powers for CRC patients. After examining the complementary effect, combined analysis of miR-223 and miR-92a, which were commonly present in stool and plasma samples, yielded the highest sensitivity of 96.8% and the specificity of 75% for CRC (AUC = 0.907). These results allowed us to establish a two-miRNA biosignature in two types of CRC clinical specimens with a high sensitivity for CRC detection.

Interaction of estrogen and progesterone in the regulation of sodium channels in collecting tubular cells.

BACKGROUND: Renal Na+ handling differs between males and females. Further, within females Na+ metabolism changes during the menstrual cycle and pregnancy. Estrogen and progesterone could regulate alpha, beta and gamma amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA levels in female rat kidney. The aim of our study is to clarify the role of the female gender steroids in the regulation of ENaC activity. METHODS: We conducted an in vitro study on cultured collecting tubular cells. The collecting tubular cells were cultured under different concentrations of estrogen and/or progesterone. We analyzed the mRNA expression and protein translation of ENaC by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Sodium channel activity was measured by short circuit current. RESULTS: Estrogen and/or progesterone alone mildly increased ENaC activity. A low concentration of estrogen together with progesterone stimulated ENaC activity more than 2-fold. A high concentration of estrogen almost completely inhibited the stimulated ENaC activity. CONCLUSION: Female gender steroid hormones affect RNA expression, protein translation and ENaC activity in renal collecting tubule cells. The effects might suggest that pre-menstruation edema is a result of subtle interaction between the female gender steroid hormones on ENaC in renal collecting tubule cells.