Releasing global forests from human management: How much more carbon could be stored?

Carbon storage in forests is a cornerstone of policy-making to prevent global warming from exceeding 1.5°C. However, the global impact of management (for example, harvesting) on the carbon budget of forests remains poorly quantified. We integrated global maps of forest biomass and management with machine learning to show that by removing human intervention, under current climatic conditions and carbon dioxide (CO2) concentration, existing global forests could increase their aboveground biomass by up to 44.1 (error range: 21.0 to 63.0) petagrams of carbon. This is an increase of 15 to 16% over current levels, equating to about 4 years of current anthropogenic CO2 emissions. Therefore, without strong reductions in emissions, this strategy holds low mitigation potential, and the forest sink should be preserved to offset residual carbon emissions rather than to compensate for present emissions levels.

Structural under-reporting of informed consent, data handling and sharing, ethical approval, and application of Open Science principles as proxies for study quality conduct in COVID-19 research: a systematic scoping review.

BACKGROUND: The COVID-19 pandemic required science to provide answers rapidly to combat the outbreak. Hence, the reproducibility and quality of conducting research may have been threatened, particularly regarding privacy and data protection, in varying ways around the globe. The objective was to investigate aspects of reporting informed consent and data handling as proxies for study quality conduct. METHODS: A systematic scoping review was performed by searching PubMed and Embase. The search was performed on November 8th, 2020. Studies with hospitalised patients diagnosed with COVID-19 over 18 years old were eligible for inclusion. With a focus on informed consent, data were extracted on the study design, prestudy protocol registration, ethical approval, data anonymisation, data sharing and data transfer as proxies for study quality. For reasons of comparison, data regarding country income level, study location and journal impact factor were also collected. RESULTS: 972 studies were included. 21.3% of studies reported informed consent, 42.6% reported waivers of consent, 31.4% did not report consent information and 4.7% mentioned other types of consent. Informed consent reporting was highest in clinical trials (94.6%) and lowest in retrospective cohort studies (15.0%). The reporting of consent versus no consent did not differ significantly by journal impact factor (p=0.159). 16.8% of studies reported a prestudy protocol registration or design. Ethical approval was described in 90.9% of studies. Information on anonymisation was provided in 17.0% of studies. In 257 multicentre studies, 1.2% reported on data sharing agreements, and none reported on Findable, Accessible, Interoperable and Reusable data principles. 1.2% reported on open data. Consent was most often reported in the Middle East (42.4%) and least often in North America (4.7%). Only one report originated from a low-income country. DISCUSSION: Informed consent and aspects of data handling and sharing were under-reported in publications concerning COVID-19 and differed between countries, which strains study quality conduct when in dire need of answers.

A cryogenic, coincident fluorescence, electron, and ion beam microscope.

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

The quest for the missing plastics: Large uncertainties in river plastic export into the sea.

Plastic pollution in the natural environment is causing increasing concern at both the local and global scale. Understanding the dispersion of plastic through the environment is of key importance for the effective implementation of preventive measures and cleanup strategies. Over the past few years, various models have been developed to estimate the transport of plastics in rivers, using limited plastic observations in river systems. However, there is a large discrepancy between the amount of plastic being modelled to leave the river systems, and the amount of plastic that has been found in the seas and oceans. Here, we investigate one of the possible causes of this mismatch by performing an extensive uncertainty analysis of the riverine plastic export estimates. We examine the uncertainty from the homogenisation of observations, model parameter uncertainty, and underlying assumptions in models. To this end, we use the to-date most complete time-series of macroplastic observations (macroplastics have been found to contain most of the plastic mass transported by rivers), coming from three European rivers. The results show that model structure and parameter uncertainty causes up to four orders of magnitude, while the homogenisation of plastic observations introduces an additional three orders of magnitude uncertainty in the estimates. Additionally, most global models assume that variations in the plastic flux are primarily driven by river discharge. However, we show that correlations between river discharge (and other environmental drivers) and the plastic flux are never above 0.5, and strongly vary between catchments. Overall, we conclude that the yearly plastic load in rivers remains poorly constrained.

Disentangling Variability in Riverbank Macrolitter Observations.

Anthropogenic macrolitter (>0.5 cm) in rivers is of increasing concern. It has been found to have an adverse effect on riverine ecosystem health, and the livelihoods of the communities depending on and living next to these ecosystems. Yet, little is known on how macrolitter reaches and propagates through these ecosystems. A better understanding of macrolitter transport dynamics is key in developing effective reduction, preventive, and cleanup measures. In this study, we analyzed a novel dataset of citizen science riverbank macrolitter observations in the Dutch Rhine-Meuse delta, spanning two years of observations on over 200 unique locations, with the litter categorized into 111 item categories according to the river-OSPAR protocol. With the use of regression models, we analyzed how much of the variation in the observations can be explained by hydrometeorology, observer bias, and location, and how much can instead be explained by temporal trends and seasonality. The results show that observation bias is very low, with only a few exceptions, in contrast with the total variance in the observations. Additionally, the models show that precipitation, wind speed, and river flow are all important explanatory variables in litter abundance variability. However, the total number of items that can significantly be explained by the regression models is 19% and only six item categories display an R2 above 0.4. This suggests that a very substantial part of the variability in macrolitter abundance is a product of chance, caused by unaccounted (and often fundamentally unknowable) stochastic processes, rather than being driven by the deterministic processes studied in our analyses. The implications of these findings are that for modeling macrolitter movement through rivers effectively, a probabilistic approach and a strong uncertainty analysis are fundamental. In turn, point observations of macrolitter need to be planned to capture short-term variability.

Accurate measurement of fast endocytic recycling kinetics in real time.

The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid, with some molecules returning to the plasma membrane with a half time <5 min. Existing methods to study these trafficking pathways utilize chemical, radioactive or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay based on a newly designed cell-impermeable fluorogenic ligand for HaloTag, Janelia Fluor 635i (JF635i, where i indicates impermeant), which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found that this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

Clathrin and clathrin adaptor AP-1 control apical trafficking of megalin in the biosynthetic and recycling routes.

Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin-Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin's apical localization and trafficking. Targeted silencing of clathrin or the Î³1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum-retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.

Vps3 and Vps8 control integrin trafficking from early to recycling endosomes and regulate integrin-dependent functions.

Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking.

Single source dual energy CT: What is the optimal monochromatic energy level for the analysis of the lung parenchyma?

OBJECTIVE: To determine the optimal monochromatic energy level for lung parenchyma analysis in spectral CT. METHODS: All 50 examinations (58% men, 64.8±16yo) from an IRB-approved prospective study on single-source dual energy chest CT were retrospectively included and analyzed. Monochromatic images in lung window reconstructed every 5keV from 40 to 140keV were independently assessed by two chest radiologists. Based on the overall image quality and the depiction/conspicuity of parenchymal lesions, each reader had to designate for every patient the keV level providing the best diagnostic and image quality. RESULTS: 72% of the examinations exhibited parenchymal lesions. Reader 1 picked the 55keV monochromatic reconstruction in 52% of cases, 50 in 30% and 60 in 18%. Reader 2 chose 50keV in 52% cases, 55 in 40%, 60 in 6% and 40 in 2%. The 50 and 55keV levels were chosen by at least one reader in 64% and 76% of all patients, respectively. Merging 50 and 55keV into one category results in an optimal setting selected by reader 1 in 82% of patients and by reader 2 in 92%, with a 74% concomitant agreement. CONCLUSION: The best image quality for lung parenchyma in spectral CT is obtained with the 50-55keV monochromatic reconstructions.

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity.

Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway.

Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum.

Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra- and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration-based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration-based washing. In the centrifugation-based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin-G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3-4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin-G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood.

The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier.

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

Gravitropism in roots of intermediate-starch mutants of Arabidopsis.

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology.

A root gravitropism mutant was isolated from the DuPont Arabidopsis thaliana T-DNA insertional mutagenesis collection. This mutant has reduced root gravitropism, hence the name rgr1. Roots of rgr1 are shorter than those of wild-type, and they have reduced lateral root formation. In addition, roots of rgr1 coil clockwise on inclined agar plates, unlike wild-type roots which grow in a wavy pattern. The rgr1 mutant has increased resistance, as measured by root elongation, to exogenously applied auxins (6-fold to indole-3-acetic acid, 3-fold to 2,4-dichlorophenoxyacetic acid, and 2-fold to napthyleneacetic acid). It is also resistant to polar auxin transport inhibitors (2-fold to triiodobenzoic acid and 3- to 5-fold to napthylphthalamic acid). The rgr1 mutant does not appear to be resistant to other plant hormone classes. When grown in the presence of 10(-7) M 2,4-dichlorophenoxyacetic acid, rgr1 roots have fewer root hairs than wild type. All these rgr1 phenotypes are Mendelian recessives. Complementation tests indicate that rgr1 is not allelic to previously characterized agravitropic or auxin-resistant mutants. The rgr1 locus was mapped using visible markers to 1.4 +/- 0.6 map units from the CH1 locus at 1-65.4. The rgr1 mutation and the T-DNA cosegregate, suggesting that rgr1 was caused by insertional gene inactivation.

DNA binding factor GT-2 from Arabidopsis.

Complementary DNA clones encoding a DNA-binding factor have been obtained from Arabidopsis by DNA hybridization with a GT-2 factor cDNA clone from rice. The GT-2 gene appears to be present as a single copy in the Arabidopsis genome and is transcribed as a 2.1 kb mRNA which is not light-regulated. The longest open reading frame in the sequenced clones predicts a protein of 65 kDa, beginning with the first in-frame methionine. The protein contains basic, acidic, and proline/glutamine-rich motifs and has significant amino acid sequence homology to the rice GT-2 factor, including three regions of 50-75 amino acids each of greater than 60% identity. Two of these regions are predicted to form similar trihelix structures postulated to be involved in selective binding to specific variations of a GT-box motif DNA sequence found in the promoter regions of several plant genes. Except for weak similarity to a tobacco GT-box binding factor, GT-1a/B2F, Arabidopsis GT-2 has no similarity to other sequences in the databases. DNA-binding studies show that Arabidopsis GT-2 has binding characteristics similar to those of the rice GT-2 factor, but dissimilar to those of the tobacco GT-1a/B2F factor. The data indicate that a DNA-binding factor containing domains of similar structure and target-sequence specificity has been conserved between monocots and dicots.

Circadian and light-regulated expression of nitrate reductase in Arabidopsis.

The expression of a number of plant genes is regulated by an endogenous circadian clock. We report that the Arabidopsis NIA2 (nitrate reductase) gene shows robust circadian oscillations in mRNA accumulation which persist for at least 5 days in plants that have been grown in a light-dark (LD) cycle and then transformed to continuous light (LL). We further show that NIA2 mRNA accumulation oscillates in a circadian fashion in plants that have been grown in LD and then transferred to continuous darkness (DD). Results from nuclear run-on transcriptional analysis suggest that the oscillations in steady-state levels of NIA2 mRNA abundance are not primarily due to changes in transcription but, instead, reflect post-transcriptional regulation. The circadian oscillations in NIA2 mRNA abundance are paralleled by circadian oscillations in nitrate reductase enzyme activity (NR activity) in Arabidopsis plants that have been grown in LD and then transferred either to DD or to LL. Etiolated Arabidopsis seedlings express neither NIA2 mRNA nor NR activity. However, both NIA2 mRNA accumulation and NR activity are induced by exposure to white light. The inductive effects of light on NIA2 mRNA accumulation are due, at least in part, to a very low fluence phytochrome-mediated response. However, the persistence of circadian oscillations in NIA2 mRNA abundance for at least 5 days in LL demonstrates that the circadian clock is capable of overriding or gating the inductive effects of light on NIA2 mRNA accumulation in Arabidopsis for an extended, continuous period of time.

Promoter and leader regions involved in the expression of the Arabidopsis ferredoxin A gene.

Ferredoxin is a nuclear-encoded protein that is involved in a variety of electron transfer reactions in both photosynthetic and non-photosynthetic plastids. We show here that the expression of the ferredoxin A gene (FedA) in Arabidopsis thaliana is light-regulated, with its mRNA level increased 4.5-fold by transfer of dark-grown seedlings to white light for 3 h. A portion of this light regulation is mediated by phytochrome through a very low fluence type of response. In addition, it is likely that another photoreceptor(s) is also involved. The FedA promoter confers a light- and tissue-regulated expression pattern when fused to the beta-glucuronidase (GUS) and luciferase reporter genes, indicating that the gene is transcriptionally regulated. No evidence of cis-acting light-regulatory elements within the 5' untranslated leader region of the gene was detected. Nevertheless, elements within this leader are required for full activity since its deletion reduces expression both in the light and dark by 25-fold. This region includes a sequence, ACAAAA, which is also present in the 5' untranslated leader of the other three ferredoxin leaders that have been sequenced and in the leaders of 31 other plant genes or about 8% of all plant genes in the GenBank database. In addition to mediating light regulation, the FedA promoter also directs GUS expression in aerial tissues at 70-fold higher levels than in roots. GUS activity staining in aerial tissues is observed in both photosynthetic and non-photosynthetic cells. These data indicate that the FedA promoter also carries the information for expression of this gene in a highly tissue- and cell-specific manner.