Detecting zoonotic and non-zoonotic pathogens in livestock and their ticks in Corsican wetlands.

BACKGROUND: Corsica is a large French island in the Mediterranean Sea with high human and animal migration rates, especially near wetlands where these migrations are particularly frequent. Among the livestock populations, cattle and sheep are widely present all across the entire Mediterranean region. Trade can be responsible for the circulation of numerous pathogens and their vectors, thereby representing a health and economic threat for the livestock industry. OBJECTIVES: The objective of our study was to investigate the presence of pathogens in cattle and sheep farms in the wetlands of Corsica using a high-throughput screening technique. METHODS: In our study, blood samples and ticks were collected from cattle and sheep in 20 municipalities near Corsican wetlands to screen for the presence of various types of pathogens. The samples were processed using a high-throughput screening technique based on real-time microfluidic PCR: 45 pathogens were screened in 47 samples simultaneously. RESULTS: A total of 372 cattle and 74 sheep were sampled, and 444 ticks were collected from cattle. Out of the eight tick species detected, the main one was Rhipicephalus bursa (38.7% of the ticks collected). From cattle blood samples, one species and two genera were found: Anaplasma marginale, Trypanosoma sp. and Babesia sp. in respectively 61.5%, 58.3% and 12.2% of the cattle blood samples. From sheep blood samples, 74.3% were positive for Anaplasma sp, 2.7% for Anaplasma ovis and 1.4% for Anaplasma capra. This is the first report of A. ovis DNA in blood samples from sheep in Corsica. Out of 444 the tick samples, 114 were positive: 77.2% for Rickettsia aeschlimannii, 20.2% for Rickettsia sp., 3.5% for Babesia sp. and 1.8% for Anaplasma sp. Among them, 2.7% were co-infected with R. aeschlimannii and Babesia sp. CONCLUSIONS: Our results confirm the extent of possible circulation of different pathogens near Corsican wetlands, not only in ticks collected from livestock but also directly in cattle and sheep, with two (Trypanosoma sp. and Babesia sp.) being detected for the first time in cattle, one for the first time in sheep (A. ovis) and one for the first time in Corsica (A. capra).

Metabolomic fingerprint of Hamamelis virginiana L. gallotannins by suspect screening analysis with UHPLC-qToF and their semiquantitative evaluation.

The evolution of the regulatory framework for medical devices in the EU (Reg 2017/745) has opened the study of complex systems emerging properties. This makes necessary to identify new analytical approaches able of characterizing complex natural substrates as completely as possible. Therefore, omics approaches and advanced analytical methods for the determination of metabolite classes appear to be at the forefront to meet this need. In this perspective, a new approach based on the suspect screening was developed to detect gallotannins. Gallotannins are a class of phenols with a polymeric nature; thus, there are no pure analytical standards available for all possible structures and their quali-quantitative determination in complex natural substrates can be a challenge. A new UHPLC-qToF method was developed and used to create an "in-house tannin database" with a dual purpose: (1) as a classic list of suspects and (2) to identify core fragments common to gallotannins to have another list of putative suspects based on the common fragment. The method was validated. The application of the method to a "system of molecules" extracted from the leaves of Hamamelis virginiana L. (Witch-hazel) allowed to the characterization of a total of 29 phenols by a suspect screening approach. Therefore, 15 gallotannins were putatively annotated while another 3 were confidently identified. All the gallotannins were semiquantified according to external regression curves of gallic acid and hamamelitannin based on core fragments at m/z 125.0244 and m/z 169.0142, the building blocks of the polymers. This new method provides a practical fit-to-purpose approach for the quali-quantitative screening evaluation of gallotannins, useful for creating multivariate control charts applicable in process development of complex natural systems or in quality control. The approach is innovative, and after specific checks, it can in principle be suitable for metabolomic fingerprint analysis of gallotannins among witch-hazel extract (WHE) samples.

Optimization and Quest of HPMC loaded Stavudine Controlled Release Dosage Development by Central Composite Design utilizing Reduced Factorial Screening Technique

Abstract The current research focused on screening and finding the significant independent variables in stavudine loaded tablet, followed by optimizing the best formulation using central composite design. The objective of the study to develop stavudine loaded controlled release tablet utilizing reduced factorial design, followed by optimization technique as well as characterization of prepared tablets. Preliminary trial batches were prepared using different grades of hydroxypropyl methylcellulose. The resolution-IV reduced factorial design was selected to screen the significant independent variables in the dosage form design. A total number of eight runs were prepared and responses were recorded. The signified factors identified by half-normal and Pareto chart. The prepared tablets are evaluated for various physiochemical characterizations. Three dependent responses such as hardness, dissolution at 6 hour and 12 hours are considered in optimization process. Later on, drug-polymer interaction study was carried out. The principal of the study design based on finding the best formulation with prefixed set parameter values utilizing the concept of screening technique. It observed that HPMC K15M (57.18 %), HPMC K100 (66.32 %) and PVP K30 (7.97 %) as best composition in a formulation batch would fulfill the predetermined parameter with specific values.

Ligand Fishing: An Approach for the Discovery of Inhibitors from Complex Biological Mixtures.

Ligand fishing is a convenient bioanalytical screening method that is based on the affinity selection of a ligand from a complex biological sample by an immobilized receptor. It is a versatile affinity-based screening approach and it has found application in multiple interacting pairs such as enzyme-inhibitor/activator, antigen-antibody, receptor-ligand, and protein-protein. Important parameters that affect the successful operation of the method are the high specificity and strong binding affinity of the interacting pair (e.g., enzyme-ligand complex) and the elution of the bound ligand from the complex. This chapter provides protocols for the synthesis of affinity adsorbent and its application in off-line ligand-fishing procedure for a 6His-tagged glutathione transferase (GST).

A Methodology for Global Sensitivity Analysis of Activated Sludge Models: Case Study with Activated Sludge Model No. 3 (ASM3).

The main objective of this study was to demonstrate a computational approach of global sensitivity analysis (GSA) integrated with functional principal component analysis (fPCA) for activated sludge models through aggregation of time-dependent model response patterns into time-independent coefficients of functional principal components (PCs). This proposed approach addresses the main issue of time-varying character of GSA indices when calculated solely on the time-dependent model outputs. The GSA-fPCA methodology was implemented using the rigorous model Activated Sludge Model No. 3 (ASM3) as case study. The approach transforms the time-dependent model outputs into functional PCs prior to calculation of GSA indices to remove the time-varying character of the calculated GSA indices. This work focused on the evaluation of the following key computational factors that may significantly influence the performance of the GSA-fPCA methodology: (a) model parameter sampling range, (b) model simulation period, (c) basis functions system, and (d) state of the system being modeled-batch or continuous activated sludge process. Results show that first few functional PCs capture up to 100% of the curve patterns in the time-dependent model outputs. The sensitivity indices calculated from the PC scores via Morris' GSA technique elucidated parameter sensitivity patterns inherent to the complex mathematical structure of ASM3. PRACTITIONER POINTS: Functional principal components-mediated GSA technique to remove time-varying character of sensitivity indices derived from time-dependent dynamical models. Technique amenable to improving efficiency of capturing response patterns into few functional principal components through various basis functions. Identifying priority parameters for ASM3 model calibration requires specification of target model outputs to which parameter sensitivities are calculated. GSA-fPCA offers a comprehensive numerical approach to manipulating models depending on the intended applications: simple fast-responding models to complex models.

Novel screening protocol for precise phenotyping of salt-tolerance at reproductive stage in rice.

The present study reports an unequivocal and improved protocol for efficient screening of salt tolerance at flowering stage in rice, which can aid phenotyping of population for subsequent identification of QTLs associated with salinity stress, particularly at reproductive stage. To validate the new method, the selection criteria, level and time of imposition of stress; plant growth medium were standardized using three rice genotypes. The setup was established with a piezometer placed in a perforated pot for continuous monitoring of soil EC and pH throughout the period of study. Further, fertilizer enriched soil was partially substituted by gravels for stabilization and maintaining the uniformity of soil EC in pots without hindering its buffering capacity. The protocol including modified medium (Soil:Stone, 4:1) at 8 dS m-1 salinity level was validated using seven different genotypes possessing differential salt sensitivity. Based on the important selection traits such as high stability index for plant yield, harvest index and number of grains/panicle and also high K+ concentration and low Na+- K+ ratio in flag leaf at grain filling stage were validated and employed in the evaluation of a mapping population in the modified screening medium. The method was found significantly efficient for easy maintenance of desired level of soil salinity and identification of genotypes tolerant to salinity at reproductive stage.

Screening of new human carbonic anhydrase Ⅱ(hCA Ⅱ) inhibitors / 中国药理学通报

Aim To provide practical method for screening human carbonic anhydrase Ⅱ(hCA Ⅱ) inhibitors in drug discovery.Methods hCA Ⅱ protein was obtained from induced BL21(DE3) E.coli containing plasmid pET-28b-hCA Ⅱ.The hCA Ⅱ activity was detected under pH 7.6 and 25℃ by its esterase activity which could decompose PNPA to increase the photoabsorption at 348 nm. After the assay conditions were finally selected, 35 new compounds were tested.Results A practical method for screening hCA Ⅱ inhibitors was successfully constituted by using recombinant hCA Ⅱ protein expressed in E.coli as the source of hCA Ⅱ enzyme.10 new compounds had better inhibitory effect and 9 new compounds had the same inhibitory effect on hCA Ⅱ compared with acetazolamide.Conclusions The hCA II inhibitor screening technique constituted in this work possesses advantages of being reliable, rapid, and practical. 19 new compounds are worth further research for developing high efficiency and low side effect drugs used for high-altitude illness.