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T cells need to adapt their cellular metabolism for effector cell differentiation. This relies on alterations in mitochondrial physiology. Which signals and molecules regulate those alterations remains unclear. We recently reported, that the mitochondrial protein TCAIM inhibits activation-induced changes in mitochondrial morphology and function and thus, CD effector T cell formation. Using conditional TCAIM knock-in (KI) and knockout (KO) mice, w now show that it also applies to CD8+ T cells and more importantly, delineate the molecular processes in mitochondria by which TCAIM controls effector cell differentiation. TCAIM KI resulted in reduced activation-induced HIF1α protein expression. Metabolomics and transcriptional data in combination with mathematical flux modeling revealed an impaired induction of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis in TCAIM KI CD8+ T cells. Addition of cholesterol completely rescued HIF1α protein expression, activation and proliferation of TCAIM KI CD8+ T cells. At the molecular level, TCAIM delayed activation-induced mitochondria-ER contact (MERC) formation by binding to MERC promoting proteins such as RMD3 and VDAC2. In summary, we demonstrate that TCAIM suppresses effector cell differentiation by inhibiting MERC formation, which induce HIF1α-mediated increase in cellular metabolism and cholesterol biosynthesis.
A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated, quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist’s experiments, while more than half have even failed to reproduce their own experiments1. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique2,3. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g., DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE4), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common Standards and guidelines for quality assessment and reproducibility5.
In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative6 was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models7,8, and tools9,10, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research.
This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of Stakeholders including, researchers, imaging scientists11, bioimage analysts, bioimage informatics developers, corporate partners, Funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREPLiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC.
To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.
Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM K55.b, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2010/2011. Nineteen national measurement institutes and the BIPM participated. Participants were required to assign the mass fraction of aldrin present as the main component in the comparison sample for CCQM-K55.b which consisted of technical grade aldrin obtained from the National Measurement Institute Australia that had been subject to serial recrystallization and drying prior to sub-division into the units supplied for the comparison.
Aldrin was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of organic compounds of medium structural complexity [molar mass range 300 Da to 500 Da] and low polarity (pKOW < -2) for which related structure impurities can be quantified by capillary gas phase chromatography (GC).
The key comparison reference value (KCRV) for the aldrin content of the material was 950.8 mg/g with a combined standard uncertainty of 0.85 mg/g. The KCRV was assigned by combination of KCRVs assigned by consensus from participant results for each orthogonal impurity class. The relative expanded uncertainties reported by laboratories having results consistent with the KCRV ranged from 0.3% to 0.6% using a mass balance approach and 0.5% to 1% using a qNMR method.
The major analytical challenge posed by the material proved to be the detection and quantification of a significant amount of oligomeric organic material within the sample and most participants relying on a mass balance approach displayed a positive bias relative to the KCRV (overestimation of aldrin content) in excess of 10 mg/g due to not having adequate procedures in place to detect and quantify the non-volatile content–specifically the non-volatile organics content–of the comparison sample.
There was in general excellent agreement between participants in the identification and the quantification of the total and individual related structure impurities, water content and the residual solvent content of the sample.
The comparison demonstrated the utility of 1H NMR as an independent method for quantitative analysis of high purity compounds. In discussion of the participant results it was noted that while several had access to qNMR estimates for the aldrin content that were inconsistent with their mass balance determination they decided to accept the mass balance result and assumed a hidden bias in their NMR data. By contrast, laboratories that placed greater confidence in their qNMR result were able to resolve the discrepancy through additional studies that provided evidence of the presence of non-volatile organic impurity at the requisite level to bring their mass balance and qNMR estimates into agreement.
The comparison required the assignment of the mass fraction of folic acid present as the main component in the comparison sample. Performance in the comparison is representative of a laboratory's measurement capability for the purity assignment of organic compounds of medium structural complexity [molecular weight range 300–500] and high polarity (pKOW < −2).
Methods used by the eighteen participating NMIs or DIs were based on a mass balance (summation of impurities) or qNMR approach, or the combination of data obtained using both methods. The qNMR results tended to give slightly lower values for the content of folic acid, albeit with larger associated uncertainties, compared with the results obtained by mass balance procedures. Possible reasons for this divergence are discussed in the report, without reaching a definitive conclusion as to their origin.
The comparison demonstrates that for a structurally complex polar organic compound containing a high water content and presenting a number of additional analytical challenges, the assignment of the mass fraction content property value of the main component can reasonably be achieved with an associated relative standard uncertainty in the assigned value of 0.5%
Results of an interlaboratory comparison on size characterization of SiO2 airborne nanoparticles using on-line and off-line measurement techniques are discussed. This study was performed in the framework of Technical Working Area (TWA) 34—'Properties of Nanoparticle Populations' of the Versailles Project on Advanced Materials and Standards (VAMAS) in the project no. 3 'Techniques for characterizing size distribution of airborne nanoparticles'. Two types of nano-aerosols, consisting of (1) one population of nanoparticles with a mean diameter between 30.3 and 39.0 nm and (2) two populations of non-agglomerated nanoparticles with mean diameters between, respectively, 36.2–46.6 nm and 80.2–89.8 nm, were generated for characterization measurements. Scanning mobility particle size spectrometers (SMPS) were used for on-line measurements of size distributions of the produced nano-aerosols. Transmission electron microscopy, scanning electron microscopy, and atomic force microscopy were used as off-line measurement techniques for nanoparticles characterization. Samples were deposited on appropriate supports such as grids, filters, and mica plates by electrostatic precipitation and a filtration technique using SMPS controlled generation upstream. The results of the main size distribution parameters (mean and mode diameters), obtained from several laboratories, were compared based on metrological approaches including metrological traceability, calibration, and evaluation of the measurement uncertainty. Internationally harmonized measurement procedures for airborne SiO2 nanoparticles characterization are proposed.
The primary crystallite size of titania powder relates to its properties in a number of applications. Transmission electron microscopy was used in this interlaboratory comparison (ILC) to measure primary crystallite size and shape distributions for a commercial aggregated titania powder. Data of four size descriptors and two shape descriptors were evaluated across nine laboratories. Data repeatability and reproducibility was evaluated by analysis of variance. One-third of the laboratory pairs had similar size descriptor data, but 83% of the pairs had similar aspect ratio data. Scale descriptor distributions were generally unimodal and were well-described by lognormal reference models. Shape descriptor distributions were multi-modal but data visualization plots demonstrated that the Weibull distribution was preferred to the normal distribution. For the equivalent circular diameter size descriptor, measurement uncertainties of the lognormal distribution scale and width parameters were 9.5% and 22%, respectively. For the aspect ratio shape descriptor, the measurement uncertainties of the Weibull distribution scale and width parameters were 7.0% and 26%, respectively. Both measurement uncertainty estimates and data visualizations should be used to analyze size and shape distributions of particles on the nanoscale.
The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the “Shed light in The daRk lineagES of the fungal tree of life” (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.
The CCQM-K148.a comparison was coordinated by the BIPM on behalf of the CCQM Organic Analysis Working Group for NMIs and DIs which provide measurement services in organic analysis under the CIPM MRA. It was undertaken as a "Track A" comparison within the OAWG strategic plan. CCQM-K148.a demonstrates capabilities for assigning the mass fraction content of a solid organic compound having moderate molecular complexity, where the compound has a molar mass in the range (75 - 500) g/mol and is non-polar (pKow < −2), when present as the primary organic component in a neat organic solid and where the mass fraction content of the primary component in the material is in excess of 950 mg/g.
Participants were required to report the mass fraction of Bisphenol A present in one supplied unit of the comparison material. Participants using a mass balance method for the assignment were also required to report their assignments of the impurity components present in the material. Methods used by the seventeen participating NMIs or DIs were predominantly based on either stand-alone mass balance (summation of impurities) or qNMR approaches, or the combination of data obtained using both methods. The results obtained using thermal methods based on freezing-point depression methods were also reported by a limited number of participants. There was excellent agreement between assignments obtained using all three approaches to assign the BPA content.
The assignment of the values for the mass fraction content of BPA consistent with the KCRV was achieved by most of the comparison participants with an associated relative standard uncertainty in the assigned value in the range (0.1 - 0.5)%.
A new facility has been developed which allows for a stable and reproducible production of ambient-like model aerosols (PALMA) in the laboratory. The set-up consists of multiple aerosol generators, a custom-made flow tube homogeniser, isokinetic sampling probes, and a system to control aerosol temperature and humidity. Model aerosols containing elemental carbon, secondary organic matter from the ozonolysis of α-pinene, inorganic salts such as ammonium sulfate and ammonium nitrate, mineral dust particles,and water were generated under different environmental conditions and at different number and mass concentrations. The aerosol physical and chemical properties were characterised with an array of experimental methods, including scanning mobility particle sizing, ion chromatography, total reflection X-ray fluorescence spectroscopy and thermo-optical analysis. The facility is very versatile and can find applications in the calibration and performance characterisation of aerosol instruments monitoring ambient air. In this study, we performed, as proof of concept, an intercomparison of three different commercial PM (particulate matter) monitors (TEOM1405, DustTrak DRX 8533 and Fidas Frog) with the gravimetric reference method under three simulated environmental scenarios. The results are presented and compared to previous field studies. We believe that the laboratory-based method for simulating ambient aerosols presented here could provide in the future a useful alternative to time-consuming and expensive field campaigns, which are often required for instrument certification and calibration.
Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi – whether transient visitors or more persistent residents – may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxo¬nomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions – such as country and host/substrate of collection – are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10–11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS bar¬code sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes – including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences – were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.