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Withdrawal StatementThe authors have withdrawn their manuscript owing to the inability to complete the revisions or follow up on the manuscript at this time, due to personal circumstances. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
O_LIIncreasing atmospheric CO2 levels have a variety of effects that can influence plant responses to microbial pathogens. However, these responses are varied, and it is challenging to predict how elevated CO2 (eCO2) will affect a particular plant-pathogen interaction. We investigated how eCO2 may influence disease development and responses to diverse pathogens in the major oilseed crop, soybean (Glycine max [L.] Merr.). C_LIO_LISoybeans grown in ambient CO2 (aCO2, 419 parts per million (ppm)) or in eCO2 (550 ppm) were challenged with bacterial, viral, fungal, and oomycete pathogens, and disease, pathogen growth, gene expression and molecular plant defense responses were quantified. C_LIO_LIIn eCO2, plants were less susceptible to Pseudomonas syringae pv. glycinea (Psg) but more susceptible to bean pod mottle virus, soybean mosaic virus, and Fusarium virguliforme. Susceptibility to Pythium sylvaticum was unchanged, although a greater loss in biomass occurred in eCO2. Reduced susceptibility to Psg was associated with enhanced defense responses. Increased susceptibility to the viruses was associated with reduced expression of antiviral defenses. C_LIO_LIThis work provides a foundation for understanding of how future eCO2 levels may impact molecular responses to pathogen challenge in soybean and demonstrates that agents infecting both shoots and roots are of potential concern in future climatic conditions. C_LI
Growth deceleration is a universal feature of growth during development: most organs and tissues slow down their growth rate much before growth termination. Using transcriptomics analysis, we show that during their two-day period of growth deceleration, wing imaginal discs of Drosophila undergo a progressive metabolic shift away from oxidative phosphorylation and towards glycolysis. We then develop an ultra-sensitive reporter HIF-1 activity, which reveals that imaginal discs become increasingly hypoxic during development in normoxic conditions, suggesting that limiting oxygen supply could underlie growth deceleration. Growth is energetically expensive and thus expected to contribute, indirectly, to oxygen consumption. Indeed, excess TOR signalling, a key stimulator of growth, triggers hypoxia locally and systemically, highlighting the need to rein in growth when oxygen becomes limiting. This is achieved by a negative feedback loop whereby the classic TOR-inhibitory function of HIF-1 is deployed in response to developmental hypoxia. The absence of Sima/HIF-1 leads to cellular stress, which is alleviated by reduced TOR signalling. Conversely, a small increase in oxygen supply reduces the stress induced by excess TOR activity. We conclude that mild hypoxia is a normal feature of organ development and that Sima/HIF-1 prevents growth-induced oxygen demand from exceeding supply.
Actin is an evolutionarily conserved cytoskeletal protein with crucial roles in cell polarity, division, migration, and muscle contraction. Actin function is regulated in part by posttranslational modifications. One such modification in non-muscle cells is arginylation, in which an arginine residue is added to the N-terminus of {beta}-actin. What is the structure of arginylated {beta}-actin (R-{beta}-actin), are its interactions with other proteins altered and what phenotypes result when R-{beta}-actin is the sole actin isoform present in the cell? Here we report the 4.2 [A] structure of ADP-bound human R-{beta}-actin filaments, the overall structure of which is nearly identical to the filaments made of non-arginylated actin. In vitro functional assays using isoform-pure actins with defined post-translational modifications reveal that the interaction between myosin-II and actin is altered upon actin arginylation, due to frequent detachment of myosin-II from R-actin filaments. In vivo, we find that replacement of the only actin gene in Schizosaccharomyces pombe with a synthetic gene encoding R-Sp-actin reduces Arp2/3-based actin patches while thickening the formin-induced actin. Furthermore, consistent with altered interactions between myosin-II and R-actin filaments, the assembly and constriction of cytokinetic actomyosin ring are perturbed in the R-Sp-actin cells. Thus, despite the overall structural similarity of arginylated and non-arginylated actin filaments, actin arginylation affects actin filament assortment into distinct subcellular structures and its interaction with myosin II.
Flavonoids represent a diverse group of plant secondary metabolites which are also discussed in context of dietary health and inflammatory response. Numerous studies have revealed that flavonoids play a central role in plant acclimation to abiotic factors like low temperature or high light, but their structural and functional diversity frequently prevents a detailed mechanistic understanding. Further complexity in analysing flavonoid metabolism arises from the different subcellular compartments which are involved in biosynthesis and storage. In the present study, non-aqueous fractionation of Arabidopsis leaf tissue was combined with metabolomics and proteomics analysis to reveal subcellular regulation during cold acclimation in two flavonoid-deficient mutants. During the first three days of a two-week cold acclimation period, flavonoid deficiency was observed to affect pyruvate, citrate and glutamate metabolism which indicated a role in stabilising C/N metabolism and photosynthesis. Also, tetrahydrofolate metabolism was found to be affected which had significant effects on regulation of the proteome of the photorespiratory pathway. In the late stage of cold acclimation, flavonoid deficiency was found to affect protein stability, folding and proteasomal degradation which resulted in a significant decrease of total protein amounts in both mutants. In summary, these findings suggest that flavonoid metabolism plays different roles in early and late stages of plant cold acclimation and significantly contributes to establishing a new protein homeostasis in a changing environment.
Coral persistence in the Anthropocene is shaped by interactions among holobiont partners (coral animals, microbial symbionts) and their environment. Cryptic coral lineages-genetically distinct yet morphologically similar groups-are critically important as they often exhibit functional diversity relevant to thermal tolerance. Additionally, environmental parameters such as thermal variability may promote tolerance, but how variability interacts with holobiont partners to shape responses to thermal challenge remains unclear. Here, we identified three cryptic lineages of Siderastrea siderea in Bocas del Toro, Panama that differ in distributions across inshore and offshore reefs, microbial associations, holobiont phenomes, and skeleton morphologies. A thermal variability experiment failed to increase thermal tolerance, but subsequent thermal challenge and recovery revealed one lineage maintained elevated energetic reserves, photochemical efficiency, and growth. Lastly, coral cores highlighted that this lineage also exhibited faster growth historically. Functional variation among cryptic lineages highlights their importance in predicting coral reef responses to climate change. TeaserCryptic host diversity drives coral phenotypes relevant to climate change.
Growth rate is a key prokaryotic trait that allows estimating fitness and understanding cell metabolism. While in some clades it has been well studied in model organisms, there is little data on slow-growing bacteria. In particular, there is a lack of quantitative studies on the species of the Brucella genus. This is an important microorganism since it is the causative agent of brucellosis, one of the most widespread bacterial zoonoses affecting several species of animals, including humans. Brucella species exhibit approximately 97% genomic similarity. Despite this, Brucella species show different host preferences, zoonotic risks, and pathogenicity. After more than one hundred years of research, numerous aspects of Brucella biology, such as in vivo and in vitro growth remain poorly characterized. In this work, we analyzed vegetative and intracellular growth of the classical Brucella species (B. abortus 2308, B. melitensis 16M. B. suis 1330, B. ovis, and B. canis). Strikingly, each species displayed particular growth parameters in culture. Doubling time (DT) spanned from 2.7 hs-1 in B. suis to 18h-1 for B. ovis. In the context of intracellular infection of J774A.1 phagocytic cells, DT was higher, but it widely varied across species, closely correlating to the growth observed in vitro. Overall, despite high similarity of the genomes, we found species-specific growth parameters in the intracellular cell cycle.
Tillering and shade tolerance are important traits in turfgrass, influenced by environmental factors, nutrients, and hormones. Shade stress negatively affects tillering. In this study, two dwarf mutants, shadow-1 and shadow-2, developed via Gamma-ray and fast-neutron mutagenesis, respectively, showed significantly higher tillering than the wild-type under greenhouse conditions. Both mutants demonstrated shade tolerance in plant height, grass quality, and color under 85% and 95% shade conditions, while shade-induced inhibition of tillering was observed in both the mutants and the wild-type. In comparison to wild-type plants under 95% shade conditions, we observed that the cytokinin biosynthetic gene IPT8 is upregulated, while the cytokinin inactivating gene CKX2 is downregulated in shadow-1. Similarly, the GA biosynthetic genes CPS1, GA2ox3, and GA20ox1 are upregulated, while the GA inactivating gene GA20ox8 is downregulated in the shadow-1 mutant. Furthermore, the ethylene biosynthetic genes ACS and ACO are also downregulated in the shadow-1 mutant. Consistently, we observed that wild-type plants exhibit increased GA and reduced CK levels, while shadow-1 mutant plants have reduced GA but increased CK levels. This explains the shadow-1 mutants shade tolerance in terms of plant height, grass quality, and color. Conversely, the tillering inhibitor genes CRY1, MAX2, and SnRK1 are upregulated in both wild-type and shadow-1 mutant plants. Our results provide novel insights into the mechanisms behind tillering and shade tolerance in turfgrasses under shade conditions.
Methanogenesis is a key aspect of anaerobic biomass degradation, and thus of global importance. While methanogenic archaea (methanogens) are ubiquitous in anaerobic habitats, the range of substrates they utilize is very limited. Most methanogens are able to grow chemolithotrophically with H2+CO2, some clades can utilize methylated compounds and/or acetate as well. Organotrophic compounds like amino acids, lipids, nucleotides, or carbohydrates, are not known to support growth of methanogens. This inability to utilize "upstream" intermediates of anaerobic biomass degradation is remarkable considering the presence of cellular metabolism that such intermediates could feed into. Here, we addressed the question why the model methanogen Methanosarcina acetivorans, despite its gluconeogenic and glycolytic capacity, is unable to utilize glucose for methanogenesis and growth. Complementing heterologously with a glucose uptake facilitator allowed a recombinant M. acetivorans strain to convert glucose to methane at a low rate. However, growth with glucose was not observed, neither as energy source nor as carbon source. Instead, methylotrophic growth of the transgenic strain was impaired in a glucose-dependent fashion, which was aggravated when also glucokinase was heterologuously produced. Glucose-dependent growth inhibition coincided with a significant - and microscopically visible - accumulation of intracellular carbohydrate. Since the glucose-utilizing trait is rapidly lost during methylotrophic growth, accumulating growth-inhibiting metabolites probably makes methanogenic and glycolytic catabolism incompatible. Thus, extensive efforts in strain development would be required to enable direct glucose utilization for methanogenesis. ImportanceThe known range of methanogenic growth substrates is very limited and the most reduced carbon compound to support organotrophic growth of M. acetivorans is pyruvate. Yet, gluconeogenesis and glycolysis, i. e., mobilization of stored glycogen, which is known to occur in M. acetivorans makes the capacity of glycolytic methanogenesis in this organism seem feasible. Although this trait could be engineered in M. acetivorans by introducing a single gene, the finding that it could not sustain growth of the organism, not even as an anabolic carbon source, demonstrates that methanogenesis is not easily compatible with carbohydrate utilization, thus probably requiring extensive metabolic rewiring.
Grapevine naturally endures stresses like heat, drought, and hypoxia. A recent study showed very low oxygen levels inside grape berries, linked to ethanol content. Other studies have established the link between ethanol and tolerance to various stresses: heat stress, drought, and high salinity. The causes of such a tolerance are not well understood. In our study, three-week-old Gamay calli, Vitis vinifera, were characterised for their endogenous oxygen levels and endogenous ethanol concentration. Subsequently, a transcriptomic study of these cells was conducted, 6 and 24 hours after treatment with 1 mM ethanol. After 6 hours, ethanol addition led to 386 differentially expressed genes, with a notable upregulation of genes related to heat response, especially small Heat Shock Proteins (sHSPs). Further experiments showed that ethanol priming in grape cells or in Arabidopsis seedlings reduced pigment and electrolyte leakage under heat stress, respectively. This study supports the idea that ethanol priming helps protect plants against heat stress and provides a valuable RNA-seq dataset for further research into the underlying mechanisms, sHSPs playing a potentially crucial role in this adaptive response.
PurposeWe aim to implement a 3D DWI sequence and show its usage on patients with new ischemic lesions. Materials and MethodsThe proposed 3D DWI sequence was implemented by integrating second-order gradient moment nulling (M2) and cardiac motion synchronization (Sync). All data were acquired on a 3T MAGNETOM Prisma scanner (Siemens Healthcare, Erlangen, Germany) using a 64 channel head and neck coil. 21 healthy volunteers underwent 3D DWI scans at 0.9 mm isotropic resolution using four motion compensation methods for comparison: no compensation (M0), M2 only, Sync only and the proposed M2+Sync method. 2D phase variation maps with different motion compensation methods were also acquired for one subject to illustrate the mechanism of the proposed method. A ghost-to-signal ratio (GSR) and blurring index was defined and compared among the four methods with repeated measures ANOVA and Tukeys test. 3D DWI was compared with 2D DWI for ADC quantification. Image quality and ischemic lesion conspicuity were evaluated with 12 patients after endovascular treatment. >ResultsWhole brain 3D DWI was achieved at 0.9 mm isotropic resolution within 5 minutes using the proposed sequence. M2+Sync achieved the lowest level of GSR and blurring along the slice direction. ADC quantification showed no statistically significant difference between M2+Sync compared to 2D DWI. 3D DWI showed similar image quality, higher lesion conspicuity and counts compared to 2D DWI. ConclusionDirect 3D DWI can be achieved by the combination of second order gradient moment nulling and cardiac synchronization.
MYT1L is a neuronal transcription factor highly expressed in the developing and adult brain. While pathogenic MYT1L mutation causes neurodevelopmental disorders, these have not been characterized in human models of neurodevelopment. Here, we defined the consequences of pathogenic MYT1L mutation in human pluripotent stem cell-derived cortical interneurons. During differentiation, mutation reduced MYT1L expression and increased progenitor cell cycle exit and neuronal differentiation and synapse-related gene expression, morphological complexity, and synaptic puncta formation. Conversely, interneuron maturation was compromised, while variant neurons exhibited altered sodium and potassium channel activity and reduced function in electrophysiological analyses. CRISPRi-based knockdown similarly impaired interneuron differentiation and maturation, supporting loss of function-based effects. We further defined MYT1L genome-wide occupancy in interneurons and related this to the transcriptomic dysregulation resulting from MYT1L mutation, to identify direct targets that could mediate these phenotypic consequences. Together, this work delineates contributors to the etiology of neurodevelopmental disorders resulting from MYT1L mutation.
Malaria kills over 600,000 people annually, with all the clinical symptoms being caused by the blood stage of the infection. Malaria parasites invade red blood cells (RBCs), where they grow and multiply until daughter parasites egress to invade new RBCs. This cycle happens primarily in the blood circulation, bone marrow, and spleen, where parasites and RBCs are exposed to flow-generated forces. Despite this, almost all in vitro growth assays are carried out in static conditions, which are a poor mimic for the conditions that malaria parasites encounter in the body. Therefore, we systematically tested the impact of dynamic conditions created by orbital shaking platforms on parasite growth and explored the link between growth and the wall shear stress forces generated by fluid motion. For several strains of the deadliest malaria species, Plasmodium falciparum, we showed that strikingly, for any given vessel, there is a critical shaking speed at which growth rates are reduced, corresponding to when the RBCs start to aggregate in the centre of the well, before growth increases again at higher shaking speeds. The force that parasites are exposed to at this critical shaking speed corresponds cloesly with previous measurements for the forces that RBCs and parasites are exposed to in the microvasculature. During invasion, the early attachment of the parasites to RBCs is dependent on two families of attachment proteins, the Erythrocyte Binding Antigen (PfEBA) family and the Reticulocyte Binding Protein Homologue (PfRH) family, which are thought to be largely redundant in function. We used a panel of PfEBA and PfRH knock-out lines to show for the first time that several of these ligands have greater importance in high wall shear stress conditions. This both adds new understanding to the function of these ligand families, and indicates that the concept of the critical shaking speed can reveal new parasite growth phenotypes.
BackgroundNoise pollution poses a significant public health risk, with prolonged exposure to high levels of noise linked to various adverse outcomes such as annoyance, sleep disturbances, cognitive impairment, hypertension, and cardiovascular diseases. Noise-induced hearing loss (NIHL) is a prominent concern in noisy occupational settings. MethodThis study investigated NIHL among 621 male Air Force soldiers aged 18-45, exposed to intense aircraft noise. Auditory assessments included pure-tone audiometry (125 Hz to 8 kHz) to categorize hearing into normal, mild, moderate, and severe impairment. Distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) were used to evaluate cochlear and auditory nerve function. Heart rate variability (HRV) provided insights into autonomic responses. Cognitive functions were assessed through computerized tests, and blood plasma was analyzed for cardiac biomarkers, oxidative stress indicators, inflammatory markers, and neurotransmitters. Proteomic analysis used iTRAQ labeling, MudPIT, and MALDI-TOF/TOF mass spectrometry for protein quantification and identification, with validation through ELISA. ResultsThe audiometric tests revealed varying degrees of hearing impairment, with significant threshold differences at 2000, 3000, 4000, and 6000 Hz, especially pronounced at 6000 Hz. The right ear showed greater impairment, and a characteristic high-frequency notch was observed, consistent with noise exposure. Proteomic analysis indicated that NIHL is associated with oxidative stress and systemic inflammation, with differential protein expression related to hearing, coagulation, and inflammation. ConclusionThis study highlights the severe impact of aircraft noise on hearing and systemic health, demonstrating correlations between hearing impairment and biochemical markers. It emphasizes the role of oxidative stress and inflammation in NIHL development and underscores the need for effective noise management and protective measures in noisy work environments.
Microglia are brains resident immune cells known for their dynamic responses to tissue and vascular injury. Little is known about how microglial activity differs across the life-stages of early development, adulthood, and aging. Using two-photon live imaging, we show that microglia in the adult cerebral cortex exhibit highly ramified processes and relatively immobile somata under basal conditions. Their responses to injury occur over minutes and are highly coordinated neighboring microglia. In neonates, microglia are denser and more mobile but less morphologically complex. Their responses to focal laser-induced injuries of capillaries or parenchymal tissue are uncoordinated, delayed and persistent over days. In the aged brain, microglia somata remain immobile under basal conditions but their processes become less ramified. Their responses to focal injuries remain coordinated but are slower and less sensitive. These studies confirm that microglia undergo significant changes in their morphology, distribution, dynamics and response to injury across the lifespan. HIGHLIGHTSO_LIMicroglia undergo significant morphological and dynamic changes between life-stages. C_LIO_LINeonatal microglia are highly dynamic, less morphologically complex and mount delayed responses to focal injury compared to adult microglia. C_LIO_LIAged microglia are slightly less ramified and their responses to focal injury are slow and less sensitive than adult microglial. C_LIO_LIMaturation of microglial morphology in the developing cortex is disrupted by focal laser injury. C_LI O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/615212v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1ba9011org.highwire.dtl.DTLVardef@1485428org.highwire.dtl.DTLVardef@18d1f2aorg.highwire.dtl.DTLVardef@1f9086d_HPS_FORMAT_FIGEXP M_FIG C_FIG
Traumatic optic neuropathy (TON) is a common cause of irreversible blindness following head injury. TON is characterized by axon damage in the optic nerve followed by retinal ganglion cell death in the days and weeks following injury. At present, no therapeutic or surgical approach has been found to offer any benefit beyond observation alone. This is due in part to the lack of translational animal models suitable for understanding mechanisms and evaluating candidate treatments. In this study, we developed a rat model of TON in which the eye is rapidly rotated, inflicting mechanical stress on the optic nerve and leading to significant visual deficits. These functional deficits were thoroughly characterized up to one week after injury using electrophysiology and immunohistochemistry. The photopic negative response (PhNR) of the light adapted full field electroretinogram (LA ffERG) was significantly altered following injury. This correlated with increased biomarkers of retinal stress, axon disruption, and cell death. Together, this evidence suggests the utility of our model for mimicking clinically relevant TON and that the PhNR may be an early diagnostic for TON. Future studies will utilize this animal model for evaluation of candidate treatments.
Recognition of the intron branch point during spliceosome assembly is a multistep process that can influence mRNA structure and levels. A branch point sequence motif UACUAAC is variably conserved in eukaryotic genomes, but in some organisms, more than one protein can recognize it. Here, we show that SF1 and Quaking (QKI) compete for a subset of intron branch sites with the sequence ACUAA. SF1 activates exon inclusion through this sequence, but QKI represses the inclusion of alternatively spliced exons with this intron branch point sequence. Using mutant reporters derived from a natural intron with two branch site-like sequences, we find that when either branch point sequence is mutated, the other is utilized; however, when both are present, neither is used due to high-affinity binding and strong splicing repression by QKI. QKI occupancy at the dual branch site directly prevents SF1 binding and subsequent recruitment of spliceosome-associated factors. Finally, the ectopic expression of QKI in budding yeast (which lacks QKI) is lethal, at least in part due to the widespread repression of splicing. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of branch point sequences that closely mirror its high-affinity binding site.
Hibernation has evolved several times in mammals to overcome harsh winter climates and food scarcity. During hibernation, animals undergo extreme shifts in metabolic rate, heart rate, respiration, and body temperature. These changes reduce energy consumption and allow animals to survive solely on their fat reserves. Understanding the mechanisms for these extreme shifts has long been recognized as a model for translational medicine as hibernators do not exhibit the same adverse effects of extended immobility that non-hibernating mammals suffer. Though work on individual species has illuminated important mechanisms of these functional changes, the genomic basis of this phenotype remains largely unknown, and few studies have drawn on comparative work to elucidate commonalities across diverse hibernating mammals. Synthesizing both single species and comparative approaches, we use metabolomic data from active and denning black bears (Ursus americanus) to guide bioinformatic analyses of genes using tests of selection and evolutionary rate convergence across independent lineages of hibernating mammals. We identify several genes with significant signatures of selection and evolutionary rate convergence in hibernators that represent both previously known and novel genetic mechanisms of the hibernation phenotype. These data provide novel insights into the genetic basis of this adaptation and serve to direct clinical research in hibernation-based therapies.