Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F3 families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F3 sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance

Echocardiography and assessing fluid responsiveness: acoustic quantification again into the picture?

Accurate identification of fluid responsiveness has become an important issue in critically ill patients. Pulse pressure and stroke volume variation have been shown to be reliable predictors of fluid responsiveness. Apart from these two valuable techniques, echo-Doppler offers an interesting alternative for estimating the adequacy of filling. Acoustic quantification is a high-tech tool for delineating the blood-tissue interface on-screen in real time. Cannesson and coworkers utilized this technique in ventilated patients to assess stroke area changes, with the intention being to predict fluid responsiveness

The Five AhMTP1 Zinc Transporters Undergo Different Evolutionary Fates towards Adaptive Evolution to Zinc Tolerance in Arabidopsis halleri

Gene duplication is a major mechanism facilitating adaptation to changing environments. From recent genomic analyses, the acquisition of zinc hypertolerance and hyperaccumulation characters discriminating Arabidopsis halleri from its zinc sensitive/non-accumulator closest relatives Arabidopsis lyrata and Arabidopsis thaliana was proposed to rely on duplication of genes controlling zinc transport or zinc tolerance. Metal Tolerance Protein 1 (MTP1) is one of these genes. It encodes a Zn2+/H+ antiporter involved in cytoplasmic zinc detoxification and thus in zinc tolerance. MTP1 was proposed to be triplicated in A. halleri, while it is present in single copy in A. thaliana and A. lyrata. Two of the three AhMTP1 paralogues were shown to co-segregate with zinc tolerance in a BC1 progeny from a cross between A. halleri and A. lyrata. In this work, the MTP1 family was characterized at both the genomic and functional levels in A. halleri. Five MTP1 paralogues were found to be present in A. halleri, AhMTP1-A1, -A2, -B, -C, and -D. Interestingly, one of the two newly identified AhMTP1 paralogues was not fixed at least in one A. halleri population. All MTP1s were expressed, but transcript accumulation of the paralogues co-segregating with zinc tolerance in the A. halleri X A. lyrata BC1 progeny was markedly higher than that of the other paralogues. All MTP1s displayed the ability to functionally complement a Saccharomyces cerevisiæ zinc hypersensitive mutant. However, the paralogue showing the least complementation of the yeast mutant phenotype was one of the paralogues co-segregating with zinc tolerance. From our results, the hypothesis that pentaplication of MTP1 could be a major basis of the zinc tolerance character in A. halleri is strongly counter-balanced by the fact that members of the MTP1 family are likely to experience different evolutionary fates, some of which not concurring to increase zinc tolerance

A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. “Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBR®GREEN qPCR analysis based on four values: the Ct- and Tm values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, “Prime number tracing”, matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBR®GREEN qPCR methods and through application of a newly developed “prime number”-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product

Belgian rare diseases plan in clinical pathology : identification of key biochemical diagnostic tests and establishment of reference laboratories and financing conditions

BackgroundOne objective of the Belgian Rare Diseases plan is to improve patients' management using phenotypic tests and, more specifically, the access to those tests by identifying the biochemical analyses used for rare diseases, developing new financing conditions and establishing reference laboratories.MethodsA feasibility study was performed from May 2015 until August 2016 in order to select the financeable biochemical analyses, and, among them, those that should be performed by reference laboratories. This selection was based on an inventory of analyses used for rare diseases and a survey addressed to the Belgian laboratories of clinical pathology (investigating the annual analytical costs, volumes, turnaround times and the tests unavailable in Belgium and outsourced abroad). A proposal of financeable analyses, financing modalities, reference laboratories' scope and budget estimation was developed and submitted to the Belgian healthcare authorities. After its approval in December 2016, the implementation phase took place from January 2017 until December 2019.ResultsIn 2019, new reimbursement conditions have been published for 46 analyses and eighteen reference laboratories have been recognized. Collaborations have also been developed with 5 foreign laboratories in order to organize the outsourcing and financing of 9 analyses unavailable in Belgium.ConclusionsIn the context of clinical pathology and rare diseases, this initiative enabled to identify unreimbursed analyses and to meet the most crucial financial needs. It also contributed to improve patients' management by establishing Belgian reference laboratories and foreign referral laboratories for highly-specific analyses and a permanent surveillance, quality and financing framework for those tests

TcOPT3, a Member of Oligopeptide Transporters from the Hyperaccumulator Thlaspi caerulescens, Is a Novel Fe/Zn/Cd/Cu Transporter

BACKGROUND: Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator. METHODOLOGY/PRINCIPAL FINDINGS: We cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe(2+) and Zn(2+). Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu). CONCLUSIONS: Our data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation