Comparing species detection success between molecular markers in DNA metabarcoding of coastal macroinvertebrates

DNA metabarcoding has great potential to improve marine biomonitoring programs by providing a rapid and accurate assessment of species composition in zoobenthic communities. However, some methodological improvements are still required, especially regarding failed detections, primers efficiency and incompleteness of databases. Here we assessed the efficiency of two different marker loci (COI and 18S) and three primer pairs in marine species detection through DNA metabarcoding of the macrozoobenthic communities colonizing three types of artificial substrates (slate, PVC and granite), sampled between 3 and 15 months of deployment. To accurately compare detection success between markers, we also compared the representativeness of the detected species in public databases and revised the reliability of the taxonomic assignments. Globally, we recorded extensive complementarity in the species detected by each marker, with 69% of the species exclusively detected by either 18S or COI. Individually, each of the three primer pairs recovered, at most, 52% of all species detected on the samples, showing also different abilities to amplify specific taxonomic groups. Most of the detected species have reliable reference sequences in their respective databases (82% for COI and 72% for 18S), meaning that when a species was detected by one marker and not by the other, it was most likely due to faulty amplification, and not by lack of matching sequences in the database. Overall, results showed the impact of marker and primer applied on species detection ability and indicated that, currently, if only a single marker or primer pair is employed in marine zoobenthos metabarcoding, a fair portion of the diversity may be overlooked.

Combination of Mycobacterium tuberculosis RS ratio and CFU improves the ability of murine efficacy experiments to distinguish between drug treatments

Murine tuberculosis drug efficacy studies have historically monitored bacterial burden based on colony forming units of M. tuberculosis in lung homogenate. In an alternative approach, a recently described molecular pharmacodynamic marker called the RS ratio quantifies drug effect on a fundamental cellular process: ongoing ribosomal RNA synthesis. Here we evaluated the ability of different pharmacodynamic markers to distinguish between treatments in three BALB/c mouse experiments at two institutions. We confirmed that different pharmacodynamic markers measure distinct biological responses. We found that a combination of pharmacodynamic markers distinguishes between treatments better than any single marker. The combination of the RS ratio with colony forming units showed the greatest ability to recapitulate the rank order of regimen treatment-shortening activity, providing proof of concept that simultaneous assessment of pharmacodynamic markers measuring different properties will enhance insight gained from animal models and accelerate development of new combination regimens. These results suggest potential for a new era in which antimicrobial therapies are evaluated not only on culture-based measures of bacterial burden but also on molecular assays that indicate how drugs impact the physiological state of the pathogen.

Simultaneous Determination of Four Ingredients in Plantago Depressa by Single Marker

Objective. To establish a quantitative analysis of multicomponents by single marker (QAMS) method for the simultaneous determination of 4 active components such as protocatechuic acid, catechin, quercetin, and luteolin in Plantago depressa. Method. 4 active components in Plantago depressa were studied. Quercetin was used as an internal reference to establish the relative correction factors among protocatechuic acid, catechin, and luteolin and calculate the contents of each component; the results were compared with those measured by the external standard method. Results. 4 components showed a good linear relationship in their respective concentration ranges (r > 0.9995). The relative correction factors (fs/k) of protocatechuic acid, catechin, and luteolin were 1.1992, 0.8613, and 1.6069, respectively. The method had good durability. The contents of protocatechuic acid, catechin, and luteolin calculated by QAMS were not significantly different from those measured by the external standard method. Conclusion. QAMS can be used to determine the content of 4 components in Plantago depressa at the same time, and the method is simple, accurate, and can be used for quality control.

An Underwater Visual Navigation Method Based on Multiple ArUco Markers

Underwater navigation presents crucial issues because of the rapid attenuation of electronic magnetic waves. The conventional underwater navigation methods are achieved by acoustic equipment, such as the ultra-short-baseline localisation systems and Doppler velocity logs, etc. However, they suffer from low fresh rate, low bandwidth, environmental disturbance and high cost. In the paper, a novel underwater visual navigation is investigated based on the multiple ArUco markers. Unlike other underwater navigation approaches based on the artificial markers, the noise model of the pose estimation of a single marker and an optimal algorithm of the multiple markers are developed to increase the precision of the method. The experimental tests are conducted in the towing tank. The results show that the proposed method is able to localise the underwater vehicle accurately.

RNA-guided cell targeting with CRISPR/RfxCas13d collateral activity in human cells

While single-cell sequencing has allowed rapid identification of novel cell types or states and associated RNA markers, functional studies remain challenging due to the lack of tools that are able to target specific cells based on these markers. Here we show that targeting a single marker RNA with CRISPR/RfxCas13d led to collateral transcriptome destruction in human cells, which can be harnessed to inhibit cell proliferation or to suppress cell state transition.

Novel clinical biomarkers in blood and pleural effusion for diagnosing patients with tuberculosis distinguishing from malignant tumor

Background Pleural effusion (PE) is a common manifestation of tuberculosis and malignant tumors, but it is difficult to distinguish tuberculous pleural effusion (TPE) and malignant pleural effusion (MPE), especially by non-invasive detection indicators. We aimed to find effective detection indexes in blood and PE for differentiating patients with tuberculosis from a malignant tumor. Methods 815 patients were collected who diagnosed with tuberculosis or cancer at Taihe hospital from 2014 to 2017. 717 patients were found to have PE by thoracoscopy. The clinical characteristics, patients’ blood parameters, and PE indicators information were summarized for analysis. Results The patients with MPE had higher percentages to be bloody and negative of Rivalta test in PE than patients with TPE. Then for clinical indicators, comparing specific parameters in blood, we observed 18 indicators were higher in the TPE group than in the MPE group. On contrast, 12 indicators were higher in the MPE group than in the TPE group (p < 0.01). In addition, in PE tests, we found there were 3 parameters higher in TPE and other 4 parameters higher in MPE patients group (p < 0.01). Then for clinical diagnosing practice, ROC and PCA analysis were applied. Top six relevant indicators with AUC value over 0.70 were screened out: pADA (0.90), pHsCRP (0.79), sMONp (0.75), sHsCRP (0.73), sESR (0.71), and sD-dimer (0.70). Moreover, with the Logistic regression model, a specific combination of 3 biomarkers pADA, sMONp, and sHsCRP could enhance distinguishing tuberculosis from malignant tumor patients with PE (AUC=0.944, 95% CI=0.925-0.964). For the top single marker pADA, we further analyzed its diagnostic function in patients with different group and observed it kept the high specificity and sensitivity. Conclusions The six indicators of pADA, pHsCRP, sMONp, sHsCRP, sESR and sD-dimer showed significant diagnostic value for clinicians. Further, the combination of pADA, sMONp, and sHsCRP has high accuracy for differential diagnosis for the first time. Mostly interestingly, pADA single marker maintained high specificity and sensitivity in patients with different status, which has great value for the rapid and accurate diagnosis of suspected cases.

Systematic Study on a Quantitative Analysis of Multicomponents by Single Marker (QAMS) Method for Simultaneous Determination of Eight Constituents in Pneumonia Mixture by UPLC-MS/MS

Pneumonia mixture was formulated and is available to treat children acute pneumonia and acute bronchitis in our hospital for nearly forty years, but there are few studies of its quality evaluation or control. In this paper, a new strategy for quality evaluation of pneumonia mixture was explored and verified through qualitative and quantitative analyses of multicomponents by single marker (QAMS) by UPLC-MS/MS. Baicalein was selected as an internal reference, and the relative correction factors (RCFs) and the relative retention time (RRT) of (R, S)-goitrin, amygdalin, chlorogenic acid, pseudoephedrine hydrochloride, ephedrine hydrochloride, ammonium glycyrrhizinate, and baicalin were established. The robustness and durability of the QAMS method were investigated. RCF values calculated by the average (AVG) method and linear regression (LRG) method had good repeatability and were acceptable for quantitative analysis, and the RTT combined with the exact masses of precursor and fragment ions and their abundance could be adopted for accurately positioning the chromatographic peak of the eight constituents. The consistency and feasibility of the QAMS method were verified by comparing the contents of the seven components calculated by a classic and validated external standard method (ESM) with those of the QAMS method, which reduces analytical cost and time of detection and avoids the problem of the diversity and large quantity of reference standards. The results demonstrated that the QAMS method developed in this paper could provide a new, alternative, and promising method to comprehensively and effectively determine multicomponents and control the quality of pneumonia mixture or even a group of similar medicines.

Environmental and Genetic Factors Affecting Apospory Expressivity in Diploid Paspalum rufum

In angiosperms, gametophytic apomixis (clonal reproduction through seeds) is strongly associated with polyploidy and hybridization. The trait is facultative and its expressivity is highly variable between genotypes. Here, we used an F1 progeny derived from diploid apomictic (aposporic) genotypes of Paspalum rufum and two F2 families, derived from F1 hybrids with different apospory expressivity (%AES), to analyze the influence of the environment and the transgenerational transmission of the trait. In addition, AFLP markers were developed in the F1 population to identify genomic regions associated with the %AES. Cytoembryological analyses showed that the %AES was significantly influenced by different environments, but remained stable across the years. F1 and F2 progenies showed a wide range of %AES variation, but most hybrids were not significantly different from the parental genotypes. Maternal and paternal genetic linkage maps were built covering the ten expected linkage groups (LG). A single-marker analysis detected at least one region of 5.7 cM on LG3 that was significantly associated with apospory expressivity. Our results underline the importance of environmental influence in modulating apospory expressivity and identified a genomic region associated with apospory expressivity at the diploid level.