External Quality Assessment of Reading and Interpretation of Malaria Rapid Diagnostic Tests among 1849 End-Users in the Democratic Republic of the Congo through Short Message Service (SMS)

Background

Although malaria rapid diagnostic tests (RDT) are simple to perform, they remain subject to errors, mainly related to the post-analytical phase. We organized the first large scale SMS based external quality assessment (EQA) on correct reading and interpretation of photographs of a three-band malaria RDT among laboratory health workers in the Democratic Republic of the Congo (DR Congo).

Methods and Findings

High resolution EQA photographs of 10 RDT results together with a questionnaire were distributed to health facilities in 9 out of 11 provinces in DR Congo. Each laboratory health worker answered the EQA by Short Message Service (SMS). Filled-in questionnaires from each health facility were sent back to Kinshasa. A total of 1849 laboratory health workers in 1014 health facilities participated. Most frequent errors in RDT reading were i) failure to recognize invalid (13.2–32.5% ) or negative test results (9.8–12.8%), (ii) overlooking faint test lines (4.1–31.2%) and (iii) incorrect identification of the malaria species (12.1–17.4%). No uniform strategy for diagnosis of malaria at the health facility was present. Stock outs of RDTs occurred frequently. Half of the health facilities had not received an RDT training. Only two thirds used the RDT recommended by the National Malaria Control Program. Performance of RDT reading was positively associated with training and the technical level of health facility. Facilities with RDT positivity rates >50% and located in Eastern DR Congo performed worse.

Conclusions

Our study confirmed that errors in reading and interpretation of malaria RDTs are widespread and highlighted the problem of stock outs of RDTs. Adequate training of end-users in the application of malaria RDTs associated with regular EQAs is recommended.     

Negative Priming Effect on Organic Matter Mineralisation in NE Atlantic Slope Sediments

The priming effect (PE) is a complex phenomenon which describes a modification (acceleration or retardation) in the mineralisation rate of refractory organic matter (OM) following inputs of labile material. PEs are well-studied in terrestrial ecosystems owing to their potential importance in the evolution of soil carbon stocks but have been largely ignored in aquatic systems despite the fact that the prerequisite for their occurrence, i.e. the co-existence of labile and refractory OM, is also true for sediments. We conducted stable isotope tracer experiments in continental margin sediments from the NE Atlantic (550–950 m) to study PE occurrence and intensity in relation to labile OM input. Sediment slurries were treated with increasing quantities of the ¹³C-labelled diatom Thalassiosira rotula and PE was quantified after 7, 14 and 21 days. There was a stepwise effect of diatom quantity on its mineralisation although mineralisation efficiency dropped with increasing substrate amounts. The addition of diatomaceous OM yielded a negative PE (i.e. retardation of existing sediment OM mineralisation) at the end of the experiment regardless of diatom quantity. Negative PE is often the result of preferential utilisation of the newly deposited labile material by the microbial community (“preferential substrate utilization”, PSU) which is usually observed at excessive substrate additions. The fact that PSU and the associated negative PE occurred even at low substrate levels in this study could be attributed to limited amounts of OM subject to priming in our study area (∼0.2% organic carbon [OC]) which seems to be an exception among continental slopes (typically >0.5%OC). We postulate that PEs will normally be positive in continental slope sediments and that their intensity will be a direct function of sediment OC content. More experiments with varying supply of substrate targeting C-poor vs. C-rich sediments are needed to confirm these hypotheses.     

Automatic wheat ear counting using machine learning based on RGB UAV imagery

In wheat (Triticum aestivum L) and other cereals, the number of ears per unit area is one of the main yield‐determining components. An automatic evaluation of this parameter may contribute to the advance of wheat phenotyping and monitoring. There is no standard protocol for wheat ear counting in the field, and moreover it is time consuming. An automatic ear‐counting system is proposed using machine learning techniques based on RGB (red, green, blue) images acquired from an unmanned aerial vehicle (UAV). Evaluation was performed on a set of 12 winter wheat cultivars with three nitrogen treatments during the 2017–2018 crop season. The automatic system uses a frequency filter, segmentation and feature extraction, with different classification techniques, to discriminate wheat ears in micro‐plot images. The relationship between the image‐based manual counting and the algorithm counting exhibited high levels of accuracy and efficiency. In addition, manual ear counting was conducted in the field for secondary validation. The correlations between the automatic and the manual in‐situ ear counting with grain yield were also compared. Correlations between the automatic ear counting and grain yield were stronger than those between manual in‐situ counting and GY, particularly for the lower nitrogen treatment. Methodological requirements and limitations are discussed.     

Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications

Ultra-deep sequencing (UDS) of amplicons is a major application for next-generation sequencing technologies, even more so for the 454 Genome Sequencer FLX. Especially for this application, errors that might be introduced during any of the sample processing or data analysis steps should be avoided or at least recognized, as they might lead to aberrant sequence variant calling. Since 454 pyrosequencing relies on PCR-driven target amplification, it is key to differentiate errors introduced during the amplification step from genuine minority variants. Thereto, optimal primer design is imperative because primer selection, primer dimer formation, and nonspecific binding may all affect the quality and outcome of amplicon-based deep sequencing. Also, other intrinsic PCR characteristics including amplification drift and the formation of secondary structures may influence sequencing data quality. We illustrate these phenomena using real life case studies and propose experimental and analytical evidence-based solutions for effective practice. Furthermore, because accuracy of the DNA polymerase is vital for reliable UDS results, a comparative analysis of error profiles from seven different DNA polymerases was performed and experimentally assessed in parallel by 454 sequencing. Finally, intra and interrun variability evaluation of the 454 sequencing protocol revealed highly reproducible results in amplicon-based UDS.     

CD27-deficient mice show normal NK-cell differentiation but impaired function upon stimulation

Natural killer (NK) cells are part of the first line defense against tumors, parasites and virus-infected cells. Therefore, factors that control NK-cell numbers and their function are important. CD27 is constitutively expressed on NK cells and its expression correlates with sequential phases in NK-cell development, discriminating phenotypically and functionally different subsets within the NK-cell population. Although CD27 has been described to have an important regulatory role in effector and memory T and B lymphocytes, its role in NK-cell biology remains to be addressed. In this study, we used CD27(-/-) mice to investigate the role of CD27 in NK-cell development and function, both during the resting state and upon stimulation. The results show that NK-cell numbers are not impaired in CD27(-/-) mice. Moreover, CD27(-/-) NK cells reach full phenotypic maturity, evidenced by normal expression of CD49b, CD43 and CD11b. Expression of activating receptors is unaltered, whereas expression of several inhibitory receptors is increased. Cytotoxicity and interferon-γ production by NK cells from CD27(-/-) mice in the resting state are normal. However, upon in vivo anti-CD40- or poly-I:C-mediated activation, or in vitro interleukin-15 priming plus anti-NKp46 stimulation, the absence of CD27 results in decreased cytolytic activity and cytokine production by spleen and liver NK cells. In conclusion, this study demonstrates that CD27 is dispensable for the development of functional NK cells. However, upon stimulation of NK cells, CD27 displays an important role in their activation and functionality.     

Expression of the Arabidopsis metallothionein 2b enhances arsenite sensitivity and root to shoot translocation in tobacco

We expressed the AtMT2b gene under the 35 S cauliflower mosaic virus promoter in Nicotiana tabacum (Sr1), using leaf disc transformation. Arsenite tolerance and uptake, as well as arsenite-induced phytochelatin (PC) accumulation in roots were measured in transgenic lines, and compared to untransformed (‘wild type’) tobacco. Measured after 5 days of exposure, arsenite tolerance was slightly but significantly decreased in the transgenic lines compared to wild type. The highest AtMT2b expressing line exhibited a significantly decreased arsenic accumulation in roots, but an increased accumulation in shoots, while the total amount of arsenic taken up remained unchanged, suggesting that AtMT2b expression enhanced the arsenic root to shoot transport. The same transformant line also exhibited a decreased rate of phytochelatin accumulation in the roots, but the phytochelatin-SH to As molar ratio was higher than in wild type, suggesting that the lower arsenite tolerance in the transformant lines was not due to a potential shortage of cysteine for PC synthesis, imposed by expression of the transgene.     

Differential protein–protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways

Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1‐ and LRRK2‐specific cellular processes by identifying their distinct interacting proteins. A protein microarray‐based interaction screen was performed with recombinant 3xFlag‐LRRK1 and 3xFlag‐LRRK2 and, in parallel, co‐immunoprecipitation followed by mass spectrometry was performed from SH‐SY5Y neuroblastoma cell lines stably expressing 3xFlag‐LRRK1 or 3xFlag‐LRRK2. We identified a set of LRRK1‐ and LRRK2‐specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF‐R) as a LRRK1‐specific interactor, while 14‐3‐3 proteins were LRRK2‐specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14‐3‐3 consensus binding motifs. To assess the functional relevance of these interactions, SH‐SY5Y‐LRRK1 and ‐LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14‐3‐3 binding, or with EGF, an EGF‐R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins.

     

A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.     

Model-assisted evaluation of crop load effects on stem diameter variations and fruit growth in peach

Key message

The paper identifies and quantifies how crop load influences plant physiological variables that determine stem diameter variations to better understand the effect of crop load on drought stress indicators.

Abstract

Stem diameter (D ^stem) variations have extensively been applied in optimisation strategies for plant-based irrigation scheduling in fruit trees. Two D ^stem derived water status indicators, maximum daily shrinkage (MDS) and daily growth rate (DGR), are however influenced by other factors such as crop load, making it difficult to unambiguously use these indicators in practical irrigation applications. Furthermore, crop load influences the growth of individual fruits, because of competition for assimilates. This paper aims to explain the effect of crop load on DGR, MDS and individual fruit growth in peach using a water and carbon transport model that includes simulation of stem diameter variations. This modelling approach enabled to relate differences in crop load to differences in xylem and phloem water potential components. As such, crop load effects on DGR were attributed to effects on the stem phloem turgor pressure. The effect of crop load on MDS could be explained by the plant water status, the phloem carbon concentration and the elasticity of the tissue. The influence on fruit growth could predominantly be explained by the effect on the early fruit growth stages.