Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, Stably Integrated Transgenic Elements.

In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.

The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.

Strigolactones promote plant freezing tolerance by releasing the WRKY41-mediated inhibition of CBF/DREB1 expression.

Cold stress is a major abiotic stress that adversely affects plant growth and crop productivity. The C-REPEAT BINDING FACTOR/DRE BINDING FACTOR 1 (CBF/DREB1) transcriptional regulatory cascade plays a key role in regulating cold acclimation and freezing tolerance in Arabidopsis (Arabidopsis thaliana). Here, we show that max (more axillary growth) mutants deficient in strigolactone biosynthesis and signaling display hypersensitivity to freezing stress. Exogenous application of GR245DS , a strigolactone analog, enhances freezing tolerance in wild-type plants and strigolactone-deficient mutants and promotes the cold-induced expression of CBF genes. Biochemical analysis showed that the transcription factor WRKY41 serves as a substrate for the F-box E3 ligase MAX2. WRKY41 directly binds to the W-box in the promoters of CBF genes and represses their expression, negatively regulating cold acclimation and freezing tolerance. MAX2 ubiquitinates WRKY41, thus marking it for cold-induced degradation and thereby alleviating the repression of CBF expression. In addition, SL-mediated degradation of SMXLs also contributes to enhanced plant freezing tolerance by promoting anthocyanin biosynthesis. Taken together, our study reveals the molecular mechanism underlying strigolactones promote the cold stress response in Arabidopsis.

A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes.

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.

Non-CG DNA hypomethylation promotes photosynthesis and nitrogen fixation in soybean.

Non-CG DNA methylation, a plant-specific epigenetic mark mainly regulated by chromomethylase (CMT), is known to play important roles in Arabidopsis thaliana. However, whether and to what extent non-CG DNA methylation modulates agronomic traits in crops remain to be explored. Here, we describe the consequences of non-CG DNA hypomethylation on development, seed composition, and yield in soybean (Glycine max). We created a Gmcmt mutant line lacking function of all four CMT genes. This line exhibited substantial hypomethylation of non-CG (CHG and CHH) sites. Non-CG hypomethylation enhanced chromatin accessibility and promoted or repressed the expression of hundreds of functionally relevant genes, including upregulation of GOLDEN-LIKE 10 (GmGLK10), which led to enhanced photosynthesis and, unexpectedly, improved nitrogen fixation efficiency. The Gmcmt line produced larger seeds with increased protein content. This study provides insights into the mechanisms of non-CG methylation-based epigenetic regulation of soybean development and suggests viable epigenetic strategies for improving soybean yield and nutritional value.

Max deletion destabilizes MYC protein and abrogates Eµ-Myc lymphomagenesis.

Although MAX is regarded as an obligate dimerization partner for MYC, its function in normal development and neoplasia is poorly defined. We show that B-cell-specific deletion of Max has a modest effect on B-cell development but completely abrogates Eµ-Myc-driven lymphomagenesis. While Max loss affects only a few hundred genes in normal B cells, it leads to the global down-regulation of Myc-activated genes in premalignant Eµ-Myc cells. We show that the balance between MYC-MAX and MNT-MAX interactions in B cells shifts in premalignant B cells toward a MYC-driven transcriptional program. Moreover, we found that MAX loss leads to a significant reduction in MYC protein levels and down-regulation of direct transcriptional targets, including regulators of MYC stability. This phenomenon is also observed in multiple cell lines treated with MYC-MAX dimerization inhibitors. Our work uncovers a layer of Myc autoregulation critical for lymphomagenesis yet partly dispensable for normal development.

The impact of multifactorial stress combination on reproductive tissues and grain yield of a crop plant.

Global warming, climate change, and industrial pollution are altering our environment subjecting plants, microbiomes, and ecosystems to an increasing number and complexity of abiotic stress conditions, concurrently or sequentially. These conditions, termed, "multifactorial stress combination" (MFSC), can cause a significant decline in plant growth and survival. However, the impacts of MFSC on reproductive tissues and yield of major crop plants are largely unknown. We subjected soybean (Glycine max) plants to a MFSC of up to five different stresses (water deficit, salinity, low phosphate, acidity, and cadmium), in an increasing level of complexity, and conducted integrative transcriptomic-phenotypic analysis of their reproductive and vegetative tissues. We reveal that MFSC has a negative cumulative effect on soybean yield, that each set of MFSC condition elicits a unique transcriptomic response (that is different between flowers and leaves), and that selected genes expressed in leaves or flowers of soybean are linked to the effects of MFSC on different vegetative, physiological, and/or reproductive parameters. Our study identified networks and pathways associated with reactive oxygen species, ascorbic acid and aldarate, and iron/copper signaling/metabolism as promising targets for future biotechnological efforts to augment the resilience of reproductive tissues of major crop plants to MFSC. In addition, we provide unique phenotypic and transcriptomic datasets for dissecting the mechanistic effects of MFSC on the vegetative, physiological, and reproductive processes of a crop plant.

Mutations in the genes responsible for the synthesis of furan fatty acids resolve the light-induced off-odor in soybean oil.

Soybean oil is the second most produced edible vegetable oil and is used for many edible and industrial materials. Unfortunately, it has the disadvantage of 'reversion flavor' under photooxidative conditions, which produces an off-odor and decreases the quality of edible oil. Reversion flavor and off-odor are caused by minor fatty acids in the triacylglycerol of soybean oil known as furan fatty acids, which produce 3-methyl-2,4-nonanedione (3-MND) upon photooxidation. As a solution to this problem, a reduction in furan fatty acids leads to a decrease in 3-MND, resulting in a reduction in the off-odor induced by light exposure. However, there are no reports on the genes related to the biosynthesis of furan fatty acids in soybean oil. In this study, four mutant lines showing low or no furan fatty acid levels in soybean seeds were isolated from a soybean mutant library. Positional cloning experiments and homology search analysis identified two genes responsible for furan fatty acid biosynthesis in soybean: Glyma.20G201400 and Glyma.04G054100. Ectopic expression of both genes produced furan fatty acids in transgenic soybean hairy roots. The structure of these genes is different from that of the furan fatty acid biosynthetic genes in photosynthetic bacteria. Homologs of these two group of genes are widely conserved in the plant kingdom. The purified oil from the furan fatty acid mutant lines had lower amounts of 3-MND and reduced off-odor after light exposure, compared with oil from the wild-type.

Assembly, comparative analysis, and utilization of a single haplotype reference genome for soybean.

Cultivar Williams 82 has served as the reference genome for the soybean research community since 2008, but is known to have areas of genomic heterogeneity among different sub-lines. This work provides an updated assembly (version Wm82.a6) derived from a specific sub-line known as Wm82-ISU-01 (seeds available under USDA accession PI 704477). The genome was assembled using Pacific BioSciences HiFi reads and integrated into chromosomes using HiC. The 20 soybean chromosomes assembled into a genome of 1.01Gb, consisting of 36 contigs. The genome annotation identified 48 387 gene models, named in accordance with previous assembly versions Wm82.a2 and Wm82.a4. Comparisons of Wm82.a6 with other near-gapless assemblies of Williams 82 reveal large regions of genomic heterogeneity, including regions of differential introgression from the cultivar Kingwa within approximately 30 Mb and 25 Mb segments on chromosomes 03 and 07, respectively. Additionally, our analysis revealed a previously unknown large (>20 Mb) heterogeneous region in the pericentromeric region of chromosome 12, where Wm82.a6 matches the 'Williams' haplotype while the other two near-gapless assemblies do not match the haplotype of either parent of Williams 82. In addition to the Wm82.a6 assembly, we also assembled the genome of 'Fiskeby III,' a rich resource for abiotic stress resistance genes. A genome comparison of Wm82.a6 with Fiskeby III revealed the nucleotide and structural polymorphisms between the two genomes within a QTL region for iron deficiency chlorosis resistance. The Wm82.a6 and Fiskeby III genomes described here will enhance comparative and functional genomics capacities and applications in the soybean community.

Effects of demographic history on recombination hotspots in soybean.

Recombination is the primary mechanism underlying genetic improvement in populations and allows plant breeders to create new allelic combinations for agronomic improvement. Soybean [Glycine max (L.) Merr.] has gone through multiple genetic bottlenecks that have significantly affected its genetic diversity, linkage disequilibrium, and altered allele frequencies. To investigate the impact of genetic bottlenecks on recombination hotspots in soybeans, historical recombination was studied in three soybean populations. The populations were wild soybean [Glycine soja (Sieb. and Zucc.)], landraces, and North American elite soybean cultivars that have been genotyped with the SoySNP50K BeadChip. While each population after a genetic bottleneck had an increased average haplotype block size, they did not have a significant difference in the number of hotspots between each population. Instead, the increase in observed haplotype block size is likely due to an elimination of individuals that contained historical recombination at hotspots which decreased the observed rate of recombination for the hotspot after each genetic bottleneck. Conversely, heterochromatic DNA which has an increased haplotype block size compared to euchromatic DNA had a significantly different number of hotspots but not a significant difference in the average hotspot recombination rate. Previously identified genomic motifs associated with hotspots were also associated with hotspots found in the historical populations suggesting a common mechanism. This characterization of historical recombination hotspots in soybeans provides further insights into the effect genetic bottlenecks and selection have on recombination hotspots.

Multi-omics-driven advances in the understanding of triacylglycerol biosynthesis in oil seeds.

Vegetable oils are rich sources of polyunsaturated fatty acids and energy as well as valuable sources of human food, animal feed, and bioenergy. Triacylglycerols, which are comprised of three fatty acids attached to a glycerol backbone, are the main component of vegetable oils. Here, we review the development and application of multiple-level omics in major oilseeds and emphasize the progress in the analysis of the biological roles of key genes underlying seed oil content and quality in major oilseeds. Finally, we discuss future research directions in functional genomics research based on current omics and oil metabolic engineering strategies that aim to enhance seed oil content and quality, and specific fatty acids components according to either human health needs or industrial requirements.

Rhizobia cystathionine γ-lyase-derived H2S delays nodule senescence in soybean.

Hydrogen sulphide (H2S) is required for optimal establishment of soybean (Glycine max)-Sinorhizobium fredii symbiotic interaction, yet its role in regulating the nitrogen fixation-senescence transition remains poorly understood. A S. fredii cystathionine γ-lyase (CSE) mutant deficient in H2S synthesis showed early nodule senescence characterized by reduced nitrogenase activity, structural changes in nodule cells, and accelerated bacteroid death. In parallel, the CSE mutant facilitated the generation of reactive oxygen species (ROS) and elicited antioxidant responses. We observed that H2S-mediated persulfidation of cysteine C31/C80 in ascorbate peroxidase (APX) and C32 in APX2 modulated enzyme activity, thereby participating in hydrogen peroxide (H2O2) detoxification and delaying nodule senescence. Comparative transcriptomic analysis revealed a significant up-regulation of GmMYB128, an MYB transcription factor (TF), in the CSE mutant nodules. Functional analysis through overexpression and RNAi lines of GmMYB128 demonstrated its role as a positive regulator in nodule senescence. MYB128-OE inoculated with the CSE mutant strain exhibited a reduction in nitrogenase activity and a significant increase in DD15 expression, both of which were mitigated by NaHS addition. Changes at the protein level encompassed the activation of plant defenses alongside turnover in carbohydrates and amino acids. Our results suggest that H2S plays an important role in maintaining efficient symbiosis and preventing premature senescence of soybean nodules.

COVID-19 detection from chest X-ray images using CLAHE-YCrCb, LBP, and machine learning algorithms.

BACKGROUND: COVID-19 is a disease that caused a contagious respiratory ailment that killed and infected hundreds of millions. It is necessary to develop a computer-based tool that is fast, precise, and inexpensive to detect COVID-19 efficiently. Recent studies revealed that machine learning and deep learning models accurately detect COVID-19 using chest X-ray (CXR) images. However, they exhibit notable limitations, such as a large amount of data to train, larger feature vector sizes, enormous trainable parameters, expensive computational resources (GPUs), and longer run-time. RESULTS: In this study, we proposed a new approach to address some of the above-mentioned limitations. The proposed model involves the following steps: First, we use contrast limited adaptive histogram equalization (CLAHE) to enhance the contrast of CXR images. The resulting images are converted from CLAHE to YCrCb color space. We estimate reflectance from chrominance using the Illumination-Reflectance model. Finally, we use a normalized local binary patterns histogram generated from reflectance (Cr) and YCb as the classification feature vector. Decision tree, Naive Bayes, support vector machine, K-nearest neighbor, and logistic regression were used as the classification algorithms. The performance evaluation on the test set indicates that the proposed approach is superior, with accuracy rates of 99.01%, 100%, and 98.46% across three different datasets, respectively. Naive Bayes, a probabilistic machine learning algorithm, emerged as the most resilient. CONCLUSION: Our proposed method uses fewer handcrafted features, affordable computational resources, and less runtime than existing state-of-the-art approaches. Emerging nations where radiologists are in short supply can adopt this prototype. We made both coding materials and datasets accessible to the general public for further improvement. Check the manuscript's availability of the data and materials under the declaration section for access.

Noisecut: a python package for noise-tolerant classification of binary data using prior knowledge integration and max-cut solutions.

BACKGROUND: Classification of binary data arises naturally in many clinical applications, such as patient risk stratification through ICD codes. One of the key practical challenges in data classification using machine learning is to avoid overfitting. Overfitting in supervised learning primarily occurs when a model learns random variations from noisy labels in training data rather than the underlying patterns. While traditional methods such as regularization and early stopping have demonstrated effectiveness in interpolation tasks, addressing overfitting in the classification of binary data, in which predictions always amount to extrapolation, demands extrapolation-enhanced strategies. One such approach is hybrid mechanistic/data-driven modeling, which integrates prior knowledge on input features into the learning process, enhancing the model's ability to extrapolate. RESULTS: We present NoiseCut, a Python package for noise-tolerant classification of binary data by employing a hybrid modeling approach that leverages solutions of defined max-cut problems. In a comparative analysis conducted on synthetically generated binary datasets, NoiseCut exhibits better overfitting prevention compared to the early stopping technique employed by different supervised machine learning algorithms. The noise tolerance of NoiseCut stems from a dropout strategy that leverages prior knowledge of input features and is further enhanced by the integration of max-cut problems into the learning process. CONCLUSIONS: NoiseCut is a Python package for the implementation of hybrid modeling for the classification of binary data. It facilitates the integration of mechanistic knowledge on the input features into learning from data in a structured manner and proves to be a valuable classification tool when the available training data is noisy and/or limited in size. This advantage is especially prominent in medical and biomedical applications where data scarcity and noise are common challenges. The codebase, illustrations, and documentation for NoiseCut are accessible for download at https://pypi.org/project/noisecut/ . The implementation detailed in this paper corresponds to the version 0.2.1 release of the software.

Development, synthesis and validation of improved c-Myc/Max inhibitors.

The pathophysiological foundations of various diseases are often subject to alteration through the utilization of small compounds, rendering them invaluable tools for the exploration and advancement of novel therapeutic strategies. Within the scope of this study, we meticulously curated a diverse library of novel small compounds meticulously designed to specifically target the c-Myc/Max complex. We conducted in vitro examinations of novel c-Myc inhibitors across a spectrum of cancer cell lines, including PANC1 (pancreatic adenocarcinoma), MCF7 (breast carcinoma), DU-145 (prostate carcinoma), and A549 (lung cancer). The initial analysis involved a 25 µM dose, which enabled the identification of potent anticancer compounds effective against a variety of tumour types. We identified c-Myc inhibitors with remarkable potency, featuring IC50 values as low as 1.6 µM and up to 40 times more effective than the reference molecule in diminishing cancer cell viability. Notably, c-Myc-i7 exhibited exceptional selectivity, displaying 37-fold and 59-fold preference for targeting prostate and breast cancers, respectively, over healthy cells. Additionally, we constructed drug-likeness models. This study underscores the potential for in vitro investigations of various tumour types using novel c-Myc inhibitors to yield ground-breaking and efficacious anticancer compounds.

Control of hyperpnoea and pulmonary gas exchange during prolonged exercise: The role of group III/IV muscle afferent feedback.

It remains unclear whether feedback from group III/IV muscle afferents is of continuous significance for regulating the pulmonary response during prolonged (>5 min), steady-state exercise. To elucidate the influence of these sensory neurons on hyperpnoea, gas exchange efficiency, arterial oxygenation and acid-base balance during prolonged locomotor exercise, 13 healthy participants (4 females; 21 (3) years, V ̇ O 2 max ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{max}}}}$ : 46 (8) ml/kg/min) performed consecutive constant-load cycling bouts at ∼50% (20 min), ∼75% (20 min) and ∼100% (5 min) of V ̇ O 2 max ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{max}}}}$ with intact (CTRL) and pharmacologically attenuated (lumbar intrathecal fentanyl; FENT) group III/IV muscle afferent feedback from the legs. Pulmonary responses were continuously recorded and arterial blood (radial catheter) periodically collected throughout the exercise. Pulmonary gas exchange efficiency was evaluated using the alveolar-arterial P O 2 ${{P}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ difference ( A - a D O 2 ${\mathrm{A - a}}{{D}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ ). There were no differences in any of the variables of interest between conditions before the start of the exercise. Pulmonary ventilation was up to 20% lower across all intensities during FENT compared to CTRL exercise (P < 0.001) and this hypoventilation was accompanied by an up to 10% lower arterial P O 2 ${{P}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ and a 2-4 mmHg higher P C O 2 ${{P}_{{\mathrm{C}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$ (both P < 0.001). The exercise-induced widening of A - a D O 2 ${\mathrm{A - a}}{{D}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ was up to 25% larger during FENT compared to CTRL (P < 0.001). Importantly, the differences developed within the first minute of each stage and persisted, or further increased, throughout the remainder of each bout. These findings reflect a critical and time-independent significance of feedback from group III/IV leg muscle afferents for continuously regulating the ventilatory response, gas exchange efficiency, arterial oxygenation and acid-base balance during human locomotion. KEY POINTS: Feedback from group III/IV leg muscle afferents reflexly contributes to hyperpnoea during short duration (i.e. <5 min) locomotor exercise. Whether continuous feedback from these sensory neurons is obligatory to ensure adequate pulmonary responses during steady-state exercise of longer duration remains unknown. Lumbar intrathecal fentanyl was used to attenuate the central projection of group III/IV leg muscle afferents during prolonged locomotor exercise (i.e. 45 min) at intensities ranging from 50% to 100% of V ̇ O 2 max ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{max}}}}$ . Without affecting the metabolic rate, afferent blockade compromised pulmonary ventilation and gas exchange efficiency, consistently impairing arterial oxygenation and facilitating respiratory acidosis throughout exercise. These findings reflect the time-independent significance of feedback from group III/IV muscle afferents for regulating exercise hyperpnoea and gas exchange efficiency, and thus for optimizing arterial oxygenation and acid-base balance, during prolonged human locomotion.

Myoglobin deficiency impairs maximal oxygen uptake and exercise performance: a lesson from Mb-/- mice.

Myoglobin (Mb) plays an important role at rest and during exercise as a reservoir of oxygen and has been suggested to regulate NO• bioavailability under hypoxic/acidic conditions. However, its ultimate role during exercise is still a subject of debate. We aimed to study the effect of Mb deficiency on maximal oxygen uptake ( V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ ) and exercise performance in myoglobin knockout mice (Mb-/- ) when compared to control mice (Mb+/+ ). Furthermore, we also studied NO• bioavailability, assessed as nitrite (NO2 - ) and nitrate (NO3 - ) in the heart, locomotory muscle and in plasma, at rest and during exercise at exhaustion both in Mb-/- and in Mb+/+ mice. The mice performed maximal running incremental exercise on a treadmill with whole-body gas exchange measurements. The Mb-/- mice had lower body mass, heart and hind limb muscle mass (P < 0.001). Mb-/- mice had significantly reduced maximal running performance (P < 0.001). V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ expressed in ml min-1 in Mb-/ - mice was 37% lower than in Mb+/+ mice (P < 0.001) and 13% lower when expressed in ml min-1  kg body mass-1 (P = 0.001). Additionally, Mb-/- mice had significantly lower plasma, heart and locomotory muscle NO2 - levels at rest. During exercise NO2 - increased significantly in the heart and locomotory muscles of Mb-/- and Mb+/+ mice, whereas no significant changes in NO2 - were found in plasma. Our study showed that, contrary to recent suggestions, Mb deficiency significantly impairs V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ and maximal running performance in mice. KEY POINTS: Myoglobin knockout mice (Mb-/- ) possess lower maximal oxygen uptake ( V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ ) and poorer maximal running performance than control mice (Mb+/+ ). Respiratory exchange ratio values at high running velocities in Mb-/- mice are higher than in control mice suggesting a shift in substrate utilization towards glucose metabolism in Mb-/- mice at the same running velocities. Lack of myoglobin lowers basal systemic and muscle NO• bioavailability, but does not affect exercise-induced NO2 - changes in plasma, heart and locomotory muscles. The present study demonstrates that myoglobin is of vital importance for V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ and maximal running performance as well as explains why previous studies have failed to prove such a role of myoglobin when using the Mb-/- mouse model.

An NBS-LRR protein in the Rpp1 locus negates the dominance of Rpp1-mediated resistance against Phakopsora pachyrhizi in soybean.

The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.

Root growth of monocotyledons and dicotyledons is limited by different tissues.

Plant growth and morphogenesis are determined by the mechanical properties of its cell walls. Using atomic force microscopy, we have characterized the dynamics of cell wall elasticity in different tissues in developing roots of several plant species. The elongation growth zone of roots of all species studied was distinguished by a reduced modulus of elasticity of most cell walls compared to the meristem or late elongation zone. Within the individual developmental zones of roots, there were also significant differences in the elasticity of the cell walls of the different tissues, thus identifying the tissues that limit root growth in the different species. In cereals, this is mainly the inner cortex, whereas in dicotyledons this function is performed by the outer tissues-rhizodermis and cortex. These differences result in a different behaviour of the roots of these species during longitudinal dissection. Modelling of longitudinal root dissection using measured properties confirmed the difference shown. Thus, the morphogenesis of monocotyledonous and dicotyledonous roots relies on different tissues as growth limiting, which should be taken into account when analyzing the localization of associated molecular events. At the same time, no matrix polysaccharide was found whose immunolabelling in type I or type II cell walls would predict their mechanical properties. However, assessment of the degree of anisotropy of cortical microtubules showed a striking correlation with the elasticity of the corresponding cell walls in all species studied.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.