Detection of Banana Mild Mosaic Virus in Musa In Vitro Plants: High-Throughput Sequencing Presents Higher Diagnostic Sensitivity Than (IC)-RT-PCR and Identifies a New Betaflexiviridae Species

: The banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus that infects Musa spp. and has a very wide geographical distribution. The current BanMMV indexing process for an accession requires the testing of no less than four plants cultivated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. We evaluated the sensitivity of different protocols for BanMMV detection from in vitro plants to accelerate the testing process. We first used corm tissues from 137 in vitro plants and obtained a diagnostic sensitivity (DSE) of only 61% when testing four plants per accession. After thermotherapy was carried out to eliminate BanMMV infection, the meristem was recovered and further grown in vitro. The same protocol was evaluated in parallel on the corm tissue surrounding the meristem, as a rapid screening to evaluate virus therapy success, and was compared to the results obtained following the standard protocol. The obtained results showed 28% false negatives when conducting testing from corm tissues, making this protocol unsuitable in routine processes. Furthermore, RT-PCR and high-throughput sequencing (HTS) tests were applied on tissues from the base (n = 39) and the leaves (n = 36). For RT-PCR, the average DSE per sample reached 65% from either the base or leaves. HTS was applied on 36 samples and yielded 100% diagnostic specificity (DSP) and 100% DSE, whatever the sampled tissue, allowing the identification of a new Betaflexiviridae species infecting Musa. These results suggest that a reliable diagnostic of BanMMV from in vitro plants using RT-PCR or HTS technologies might represent an efficient alternative for testing after greenhouse cultivation.

Rapid screening and characterization of caffeic acid metabolites in rats by UHPLC-Q-TOF mass spectrometry

Purpose: To determine the metabolism of caffeic acid in rats. Methods: Sprague-Dawley rats were intragastrically administered caffeic acid in saline suspension, and biological samples collected. After sample pretreatment by solid phase extraction, ultra-high performance liquid chromatography combined with quadrupole-time of flight mass spectrometry system (UHPLC-Q-TOF-MS/MS) was established to rapidly screen and characterize caffeic acid metabolites in rats. Waters HSS T3 UPLC chromatographic column (2.1 mm × 100 mm, 1.7 μm) was applied for the gradient elution with aqueous solution of formic acid (A)-acetonitrile (B). Mass spectral data for the biological samples in electrospray positive and negative ion modes were collected and analyzed by SCIEX OS 1.3 workstation. Results: Based on their precise molecular weights and multistage mass spectrometry cleavage information, caffeic acid and 21 metabolites in vivo were identified. The results demonstrate that the biotransformation of caffeic acid in vivo was mainly achieved via hydrogenation, hydroxylation, methylation, sulfonation, glucuronidation, acetylation, and composite reactions. Conclusion: The metabolites and metabolic pathways of caffeic acid in rats have been rapidly elucidated, and its potential pharmacodynamics forms have been clarified. This provides a valuable and meaningful reference for the study of caffeic acid metabolites, biological activities, and its medicinal material basis in vivo.

Morphological and Physiological Screening to Predict Lettuce Biomass Production in Controlled Environment Agriculture

Fast growth and rapid turnover is an important crop trait in controlled environment agriculture (CEA) due to its high cost. An ideal screening approach for fast-growing cultivars should detect desirable phenotypes non-invasively at an early growth stage, based on morphological and/or physiological traits. Hence, we established a rapid screening protocol based on a simple chlorophyll fluorescence imaging (CFI) technique to quantify the projected canopy size (PCS) of plants, combined with electron transport rate (ETR) measurements using a chlorophyll fluorometer. Eleven lettuce cultivars (Lactuca sativa), selected based on morphological differences, were grown in a greenhouse and imaged twice a week. Shoot dry weight (DW) of green cultivars at harvest 51 days after germination (DAG) was correlated with PCS at 13 DAG (R2 = 0.74), when the first true leaves had just appeared and the PCS was <8.5 cm2. However, early PCS of high anthocyanin (red) cultivars was not predictive of DW. Because light absorption by anthocyanins reduces the amount of photons available for photosynthesis, anthocyanins lower light use efficiency (LUE; DW/total incident light on canopy over the cropping cycle) and reduce growth. Additionally, the total incident light on the canopy throughout the cropping cycle explained 90% and 55% of variability in DW within green and red cultivars, respectively. Estimated leaf level ETR at a photosynthetic photon flux density (PPFD) of 200 or 1000 µmol m−2 s−1 were not correlated with DW in either green or red cultivars. In conclusion, early PCS quantification is a useful tool for the selection of fast-growing green lettuce phenotypes. However, this approach may not work in cultivars with high anthocyanin content because anthocyanins direct excitation energy away from photosynthesis and growth, weakening the correlation between incident light and growth.

Comparative Assessment of Antibiotic Residues Using Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS) and a Rapid Screening Test in Raw Milk Collected from the North-Central Algerian Dairies

Antibiotic residues in milk are a major health threat for the consumer and a hazard to the dairy industry, causing significant economic losses. This study aims to assess the presence of antibiotic residues in raw milk comparatively by a rapid screening test (BetaStar® Combo) and Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). A total of 445 samples were collected from 3 dairy companies of north-central Algeria (Algiers, Blida, Boumerdes), and they were rapidly screened for β-lactams and tetracyclines; 52 samples, comprising 34 positive tanker-truck milk and 18 negative bulk-tank milk were tested by LC-MS/MS, which revealed 90.4% were contaminated (n = 47) and 55.3% exceeded the Maximum Residue Limit (MRL). The β-lactams as parent compounds and their metabolites were the most frequently detected with maximum value for cloxacillin (1231 µg/kg) and penicillin G (2062 µg/kg). Under field condition, the false-positive results, particularly for tetracyclines, seems to be related to milk samples displaying extreme acidity values (≥19°D) or fat-level fluctuations (2.7 g/100 mL and 5.6–6.2 g/100 mL). Despite a relatively low prevalence (7.64%) of residues using the rapid test, the detection by LC-MS/MS of flumequine (52 µg/kg), cefaclor (maximum 220 µg/kg) and metabolites of β-lactams at high levels should lead to reflections on the control of their human and environmental toxicological effects.

Cell-based screen identifies human type I interferon-stimulated regulators of Toxoplasma gondii Infection

Macrophages activated with interferons (IFNs) respond with transcriptional changes that enhance clearance of intracellular pathogens such as Toxoplasma, a ubiquitous apicomplexan parasite that infects more than a billion people worldwide. Although IFNs generally inhibit Toxoplasma, the parasite can also induce components of the host IFN signalling pathway to enhance survival in host cells. Compared to the type II IFN gamma (IFNγ), the role of type I IFNs in macrophage response to Toxoplasma is relatively not well characterized. Here, using fluorescent Toxoplasma and a CRISPR/Cas9 knockout library that only targets interferon-stimulated genes (ISGs), we adapted a loss-of-function flow cytometry-based approach to systematically identify type I ISGs that control Toxoplasma growth in THP-1 cells, a human macrophage cell line. The system enabled the rapid screening of more than 1900 ISGs for type I (IFNα)-induced inhibitors and enhancers of Toxoplasma growth in THP-1 cells. We identified 26 genes that are associated with Toxoplasma growth arrest out of which we confirmed MAX, SNX5, F2RL2, and SSB, as potent IFNα-induced inhibitors of Toxoplasma in THP1 cells. These findings provide a genetic and experimental roadmap to elucidate type I IFN-induced cell-autonomous responses to Toxoplasma.