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Sorghum bicolor (sorghum) is a vital C4 monocotyledon crop cultivated in arid regions worldwide, valued for its significance in both human and animal nutrition. Despite its agricultural prominence, sorghum research has been hindered by low transformation frequency. In this study, we examined sorghum transformation using the pVS1-VIR2 ternary vector system for Agrobacterium, combined with the morphogenic genes BABY BOOM and WUSCHEL2 and selection using G418. We optimized Agrobacterium-mediated infection, targeting key parameters such as bacterial optical density, co-cultivation time, and temperature. Additionally, an excision-based transformation system enabled us to generate transgenic plants free of morphogenic regulators. The method yielded remarkable transformation frequencies, reaching up to 164.8% based on total isolated plantlets. The same combination of ternary vector, morphogenic genes and geneticin-based selection also resulted in a marked increase in transformation efficiency of the Zea mays (maize) inbred line B104. The potential for genomic editing using this approach positions it as a valuable tool for the development of sorghum and maize varieties that comply with evolving European regulations. Our work marks a significant stride in sorghum biotechnology and holds promise for addressing global food security challenges in a changing climate., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Motor skill learning, the process of acquiring new motor skills, is critically important across the lifespan, from early development through adulthood and into older age, as well as in pathological conditions (i.e., rehabilitation). Extensive research has demonstrated that motor skill acquisition in young adults is accompanied by significant neuroplastic changes, including alterations in brain structure (gray and white matter), function (i.e., activity and connectivity), and neurochemistry (i.e., levels of neurotransmitters). In the aging population, motor performance typically declines, characterized by slower and less accurate movements. However, despite these age-related changes, older adults maintain the capacity for skill improvement through training. In this review, we explore the extent to which the aging brain retains the ability to adapt in response to motor learning, specifically whether skill acquisition is accompanied by neural changes. Furthermore, we discuss the associations between inter-individual variability in brain structure and function and the potential for future learning in older adults. Finally, we consider the use of non-invasive brain stimulation techniques aimed at optimizing motor learning in this population. Our review provides insights into the neurobiological underpinnings of motor learning in older adults and emphasizes strategies to enhance their motor skill acquisition., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J. Gooijers reports financial support was provided by Research Fund KU Leuven. S.P. Swinnen reports financial support was provided by Research Foundation Flanders. S.P. Swinnen reports financial support was provided by Excellence of Science. M. Hehl reports financial support was provided by Research Foundation Flanders. C. Seer reports financial support was provided by Research Foundation Flanders. M. Hehl reports financial support was provided by Research Fund KU Leuven. C. Seer reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. S.P. Swinnen reports financial support was provided by Research Fund KU Leuven. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Drought is one of the most devastating causes of yield losses in crops like maize, and the anticipated increases in severity and duration of drought spells due to climate change pose an imminent threat to agricultural productivity. To understand the drought response, phenotypic and molecular studies are typically performed at a given time point after drought onset, representing a steady-state adaptation response. Because growth is a dynamic process, we monitored the drought response with high temporal resolution and examined cellular and transcriptomic changes after rehydration at 4 and 6 days after leaf four appearance. These data showed that division zone activity is a determinant for full organ growth recovery upon rehydration. Moreover, a prolonged maintenance of cell division by the ectopic expression of PLASTOCHRON1 extends the ability to resume growth after rehydration. The transcriptome analysis indicated that GROWTH-REGULATING FACTORS (GRFs) affect leaf growth by impacting cell division duration, which was confirmed by a prolonged recovery potential of the GRF1-overexpression line after rehydration. Finally, we used a multiplex genome editing approach to evaluate the most promising differentially expressed genes from the transcriptome study and as such narrowed down the gene space from 40 to seven genes for future functional characterization., (© 2024 John Wiley & Sons Ltd.)

Background: Hypocholesterolemia hallmarks critical illness though the underlying pathophysiology is incompletely understood. As low circulating cholesterol levels could partly be due to an increased conversion to cortisol/corticosterone, we hypothesized that glucocorticoid treatment, via reduced de novo adrenal cortisol/corticosterone synthesis, might improve cholesterol availability and as such affect adrenal gland and skeletal muscle function., Methods: In a matched set of prolonged critically ill patients (n = 324) included in the EPaNIC RCT, a secondary analysis was performed to assess the association between glucocorticoid treatment and plasma cholesterol from ICU admission to day five. Next, in a mouse model of cecal ligation and puncture-induced sepsis, septic mice were randomized to receive either hydrocortisone (1.2 mg/day) (n = 17) or placebo (n = 15) for 5 days, as compared with healthy mice (n = 18). Plasma corticosterone, cholesterol, and adrenocortical and myofiber cholesterol were quantified. Adrenal structure and steroidogenic capacity were evaluated. Muscle force and markers of atrophy, fibrosis and regeneration were quantified. In a consecutive mouse study with identical design (n = 24), whole body composition was assessed by EchoMRI to investigate impact on lean mass, fat mass, total and free water., Results: In human patients, glucocorticoid treatment was associated with higher plasma HDL- and LDL-cholesterol from respectively ICU day two and day three, up to day five (P < 0.05). Plasma corticosterone was no longer elevated in hydrocortisone-treated septic mice compared to placebo, whereas the sepsis-induced reduction in plasma HDL- and LDL-cholesterol and in adrenocortical cholesterol was attenuated (P < 0.05), but without improving the adrenocortical ACTH-induced CORT response and with increased adrenocortical inflammation and apoptosis (P < 0.05). Total body mass was further decreased in hydrocortisone-treated septic mice (P < 0.01) compared to placebo, with no additional effect on muscle mass, force or myofiber size. The sepsis-induced rise in markers of muscle atrophy and fibrosis was unaffected by hydrocortisone treatment, whereas markers of muscle regeneration were suppressed compared to placebo (P < 0.05). An increased loss of lean body mass and total and free water was observed in hydrocortisone-treated septic mice compared to placebo (P < 0.05)., Conclusions: Glucocorticoid treatment partially attenuated critical illness-induced hypocholesterolemia, but at a cost of impaired adrenal function, suppressed muscle regeneration and exacerbated loss of body mass., (© 2024. The Author(s).)

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

The recalcitrance exhibited by many maize ( Zea mays ) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium -mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER , which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hernandes-Lopes, Pinto, Vieira, Monteiro, Gerasimova, Nonato, Bruno, Vikhorev, Rausch-Fernandes, Gerhardt, Pauwels, Arruda, Dante and Yassitepe.)

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.

The HD-ZIP III transcription factor REVOLUTA (REV) is involved in early leaf development, as well as in leaf senescence. REV directly binds to the promoters of senescence-associated genes, including the central regulator WRKY53 . As this direct regulation appears to be restricted to senescence, we aimed to characterize protein-interaction partners of REV which could mediate this senescence-specificity. The interaction between REV and the TIFY family member TIFY8 was confirmed by yeast two-hybrid assays, as well as by bimolecular fluorescence complementation in planta. This interaction inhibited REV's function as an activator of WRKY53 expression. Mutation or overexpression of TIFY8 accelerated or delayed senescence, respectively, but did not significantly alter early leaf development. Jasmonic acid (JA) had only a limited effect on TIFY8 expression or function; however, REV appears to be under the control of JA signaling. Accordingly, REV also interacted with many other members of the TIFY family, namely the PEAPODs and several JAZ proteins in the yeast system, which could potentially mediate the JA-response. Therefore, REV appears to be under the control of the TIFY family in two different ways: a JA-independent way through TIFY8, which controls REV function in senescence, and a JA-dependent way through PEAPODs and JAZ proteins.

Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBE RecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBE RecAp -A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria., (© 2023. The Author(s).)

Context: Muscle expresses and secretes several myokines that bring about benefits in distant organs., Objective: We investigated the impact of critical illness on muscular expression of irisin, kynurenine aminotransferases, and amylase; association with clinical outcome; and impact of interventions that attenuate muscle wasting/weakness., Methods: We studied critically ill patients who participated in 2 randomized controlled trials (EPaNIC/NESCI) and documented time profiles in critically ill mice. Included in the study were 174 intensive care unit (ICU) patients (day 8 ± 1) vs 19 matched controls, and 60 mice subjected to surgery/sepsis vs 60 pair-fed healthy mice. Interventions studied included 7-day neuromuscular electrical stimulation (NMES), and withholding parenteral nutrition (PN) in the first ICU week (late PN) vs early PN. The main outcome measures were FNDC5 (irisin- precursor), KYAT1, KYAT3 , and amylase mRNA expression in skeletal muscle., Results: Critically ill patients showed 34% to 80% lower mRNA expression of FNDC5 , KYAT1 , and amylases than controls ( P < .0001). Critically ill mice showed time-dependent reductions in all mRNAs compared with healthy mice ( P ≤ .04). The lower FNDC5 expression in patients was independently associated with a higher ICU mortality ( P = .015) and ICU-acquired weakness ( P = .012), whereas the lower amylase expression in ICU survivors was independently associated with a longer ICU stay ( P = .0060). Lower amylase expression was independently associated with a lower risk of death ( P = .048), and lower KYAT1 expression with a lower risk of weakness ( P = .022). NMES increased FNDC5 expression compared with unstimulated muscle ( P = .016), and late PN patients had a higher KYAT1 expression than early PN patients ( P = .022)., Conclusion: Expression of the studied myokines was affected by critical illness and associated with clinical outcomes, with limited effects of interventions that attenuate muscle wasting or weakness., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The characterization of callosal white matter is crucial for understanding the relationship between brain structure and bimanual motor function. An earlier literature review established this. With advancements in neuroimaging and data modeling, we aim to provide an update on the existing literature. Firstly, we highlight new CC parcellation approaches, such as functional MRI- and atlas-informed tractography and in vivo histology. Secondly, we elaborate on recent insights into the CC's role in bimanual coordination, drawing evidence from studies on healthy young and older adults, patients and training-related callosal plasticity. We also reflect on progress in the field and propose future perspectives to inspire research on the underlying mechanisms of structural-functional interactions.

Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Modern agriculture is struggling to meet the increasing food, silage and raw material demands due to the rapid growth of population and climate change. In Arabidopsis, DA1 and DAR1 are proteases that negatively regulate cell proliferation and control organ size. DA1 and DAR1 are activated by ubiquitination catalyzed by the E3 ligase BIG BROTHER (BB). Here, we characterized the DA1, DAR1 and BB gene families in maize and analyzed whether perturbation of these genes regulates organ size similar to what was observed in Arabidopsis. We generated da1&#95;dar1a&#95;dar1b triple CRISPR maize mutants and bb1&#95;bb2 double mutants. Detailed phenotypic analysis showed that the size of leaf, stem, cob, and seed was not consistently enlarged in these mutants. Also overexpression of a dominant-negative DA1 R333K allele, resembling the da1-1 allele of Arabidopsis which has larger leaves and seeds, did not alter the maize phenotype. The mild negative effects on plant height of the DA1 R333K &#95;bb1&#95;bb2 mutant indicate that the genes in the DA1 pathway may control organ size in maize, albeit less obvious than in Arabidopsis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Plant flowers have a functional life span during which pollination and fertilization occur to ensure seed and fruit development. Once flower senescence is initiated, the potential to set seed or fruit is irrevocably lost. In maize, silk strands are the elongated floral stigmas that emerge from the husk-enveloped inflorescence to intercept airborne pollen. Here we show that KIRA1-LIKE1 (KIL1), an ortholog of the Arabidopsis NAC (NAM (NO APICAL MERISTEM), ATAF1/2 (Arabidopsis thaliana Activation Factor1 and 2) and CUC (CUP-SHAPED COTYLEDON 2)) transcription factor KIRA1, promotes senescence and programmed cell death (PCD) in the silk strand base, ending the window of accessibility for fertilization of the ovary. Loss of KIL1 function extends silk receptivity and thus strongly increases kernel yield following late pollination. This phenotype offers new opportunities for possibly improving yield stability in cereal crops. Moreover, despite diverging flower morphologies and the substantial evolutionary distance between Arabidopsis and maize, our data indicate remarkably similar principles in terminating floral receptivity by PCD, whose modulation offers the potential to be widely used in agriculture., (� American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In septic mice, 3-hydroxybutyrate-sodium-salt has shown to partially prevent sepsis-induced muscle weakness. Although effective, the excessive sodium load was toxic. We here investigated whether ketone ester 3-hydroxybutyl-3-hydroxybutanoate (3HHB) was a safer alternative. In a mouse model of abdominal sepsis, the effects of increasing bolus doses of 3HHB enantiomers on mortality, morbidity and muscle force were investigated (n = 376). Next, plasma 3HB - clearance after bolus D-3HHB was investigated (n = 27). Subsequently, in septic mice, the effect on mortality and muscle force of a continuous D,L-3HHB infusion was investigated (n = 72). In septic mice, as compared with placebo, muscle force was increased at 20 mmol/kg/day L-3HHB and at 40 mmol/kg/day D- and D,L-3HHB. However, severity of illness and mortality was increased by doubling the effective bolus doses. Bolus 3HHB caused a higher 3HB - plasma peak and slower clearance with sepsis. Unlike bolus injections, continuous infusion of D,L-3HHB did not increase severity of illness or mortality, while remaining effective in improving muscle force. Treatment of septic mice with the ketone ester 3HHB partly prevented muscle weakness. Toxicity of 3HHB administered as bolus was completely avoided by continuous infusion of the same dose. Whether continuous infusion of ketone esters represents a promising intervention to also prevent ICU-acquired weakness in human patients should be investigated., (© 2022. The Author(s).)

Background: Reduced glucocorticoid-receptor (GR) expression in blood suggested that critically ill patients become glucocorticoid-resistant necessitating stress-doses of glucocorticoids. We hypothesised that critical illness evokes a tissue-specific, time-dependent expression of regulators of GR-action which adaptively guides glucocorticoid action to sites of need., Methods: We performed a prospective, observational, cross-sectional human study and two translational mouse studies. In freshly-isolated neutrophils and monocytes and in skeletal muscle and subcutaneous adipose tissue of 137 critically ill patients and 20 healthy controls and in skeletal muscle and adipose tissue as well as in vital tissues (heart, lung, diaphragm, liver, kidney) of 88 septic and 26 healthy mice, we quantified gene expression of cortisone-reductase 11β-HSD1, glucocorticoid-receptor-isoforms GRα and GRβ, GRα-sensitivity-regulating-co-chaperone FKBP51, and GR-action-marker GILZ. Expression profiles were compared in relation to illness-duration and systemic-glucocorticoid-availability., Findings: In patients' neutrophils, GRα and GILZ were substantially suppressed (p≤0·05) throughout intensive care unit (ICU)-stay, while in monocytes low/normal GRα coincided with increased GILZ (p≤0·05). FKBP51 was increased transiently (neutrophils) or always (monocytes,p≤0·05). In patients' muscle, 11β-HSD1 and GRα were low-normal (p≤0·05) and substantially suppressed in adipose tissue (p≤0·05); FKBP51 and GILZ were increased in skeletal muscle (p≤0·05) but normal in adipose tissue. GRβ was undetectable. Increasing systemic glucocorticoid availability in patients independently associated with further suppressed muscle 11β-HSD1 and GRα, further increased FKBP51 and unaltered GILZ (p≤0·05). In septic mouse heart and lung, 11β-HSD1, FKBP51 and GILZ were always high (p≤0·01). In heart, GRα was suppressed (p≤0·05), while normal or high in lung (all p≤0·05). In diaphragm, 11β-HSD1 was high/normal, GRα low/normal and FKBP51 and GILZ high (p≤0·01). In kidney, 11β-HSD1 transiently increased but decreased thereafter, GRα was normal and FKBP51 and GILZ high (p≤0·01). In liver, 11β-HSD1 was suppressed (p≤0·01), GRα normal and FKBP51 high (p≤0·01) whereas GILZ was transiently decreased but elevated thereafter (p≤0·05). Only in lung and diaphragm, treatment with hydrocortisone further increased GILZ., Interpretation: Tissue-specific, time-independent adaptations to critical illness guided GR-action predominantly to vital tissues such as lung, while (partially) protecting against collateral harm in other cells and tissues, such as neutrophils. These findings argue against maladaptive generalised glucocorticoid-resistance necessitating glucocorticoid-treatment., Funding: Research-Foundation-Flanders, Methusalem-Program-Flemish-Government, European-Research-Council, European-Respiratory-Society., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

This paper analyses the lived experiences of people living with HIV in South Africa through the use of body mapping as a visual research method, by focusing on the physical and symbolic use of the body within the broader context of anthropology and medical anthropology. The study consists of an empirical analysis of the body maps themselves and the accompanied narratives of seven participants, six female and one male participant living with HIV in South Africa. Drawing upon theories and literature on theorising the body in medical anthropology and visual research, this study explores the significance of this practice as a visual research method in understanding the nuanced lived experiences of people living with HIV by highlighting the individuality of the body and emotions; embodied experiences: a bio-cultural approach; and the body politic: social injustice. The results of this study illustrate that body mapping is a unique visual research method, that explores the body as the vehicle in which we exist within the world, while containing a vast amount of layered interpretive and cultural meanings, which are key to understanding the lived experience of people from marginalised groups.

Background: Intensive care unit (ICU)-acquired weakness can persist beyond ICU stay and has been associated with long-term functional impairment of ICU survivors. Recently, DNA methylation alterations were found in the blood of ICU patients, partially explaining long-term developmental impairment of critically ill children. As illness-induced aberrant DNA methylation theoretically could also be involved in long-term weakness, we investigated whether the DNA methylation signature in muscle of adult critically ill patients differs from that in muscle of healthy controls., Methods: Genome-wide methylation was determined (Infinium® HumanMethylationEPIC BeadChips) in DNA extracted from skeletal muscle biopsies that had been collected on Day 8 ± 1 in ICU from 172 EPaNIC-trial patients [66% male sex, median age 62.7 years, median body mass index (BMI) 25.9 kg/m 2 ] and 20 matched healthy controls (70% male sex, median age 58.0 years, median BMI 24.4 kg/m 2 ). Methylation status of individual cytosine-phosphate-guanine (CpG) sites of patients and controls was compared with F-tests, using the Benjamini-Hochberg false discovery rate to correct for multiple comparisons. Differential methylation of DNA regions was assessed with bump hunting, with 1000 permutations assessing uncertainty, expressed as family-wise error rate. Gene expression was investigated for 10 representative affected genes., Results: In DNA from ICU patients, 565 CpG sites, associated with 400 unique genes, were differentially methylated as compared with controls (average difference 3.2 ± 0.1% ranging up to 16.9%, P < 0.00005). Many of the associated genes appeared highly relevant for muscle structure and function/weakness, including genes involved in myogenesis, muscle regeneration, nerve/muscle membrane excitability, muscle denervation/re-innervation, axon guidance/myelination/degeneration/regeneration, synapse function, ion channelling with especially calcium signalling, metabolism (glucose, protein, and fat), insulin signalling, neuroendocrine hormone regulation, mitochondrial function, autophagy, apoptosis, oxidative stress, Wnt signalling, transcription regulation, muscle fat infiltration during regeneration, and fibrosis. In patients as compared with controls, we also identified two hypomethylated regions, spanning 18 and 3 CpG sites in the promoters of the HIC1 and NADK2 genes, respectively (average differences 5.8 ± 0.01% and 12.1 ± 0.04%, family-wise error rate <0.05). HIC1 and NADK2 play important roles in muscle regeneration and postsynaptic acetylcholine receptors and in mitochondrial processes, respectively. Nine of 10 investigated genes containing DNA methylation alterations were differentially expressed in patients as compared with controls (P ≤ 0.03)., Conclusions: Critically ill patients present with a different DNA methylation signature in skeletal muscle as compared with healthy controls, which in theory could provide a biological basis for long-term persistence of weakness in ICU survivors., Trial Registration: ClinicalTrials.gov: NCT00512122, registered on 31 July 2007., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE ( VYL ) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aesaert, Impens, Coussens, Van Lerberge, Vanderhaeghen, Desmet, Vanhevel, Bossuyt, Wambua, Van Lijsebettens, Inzé, De Keyser, Jacobs, Karimi and Pauwels.)

CRISPR/Cas9 genome editing has been used extensively in a wide variety of plant species. Creation of loss-of-function alleles, promoter variants and mutant collections are a few of the many uses of genome editing. In a typical workflow for sexually reproducing species, plants are generated that contain an integrated CRISPR/Cas9 transgene. After editing of the gene of interest, T-DNA null segregants can be identified in the next generation that contain only the desired edit. However, maintained presence of the CRISPR/Cas9 transgene and continued editing in the subsequent generations offer a range of applications for model plants and crops. In this review, we define transgenerational gene editing (TGE) as the continued editing of CRISPR/Cas9 after a genetic cross. We discuss the concept of TGE, summarize the current main applications, and highlight special cases to illustrate the importance of TGE for plant genome editing research and breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Impens, Jacobs, Nelissen, Inzé and Pauwels.)

The plant shoot apex houses the shoot apical meristem, a highly organized and active stem-cell tissue where molecular signaling in discrete cells determines when and where leaves are initiated. We optimized a spatial transcriptomics approach, in situ sequencing (ISS), to colocalize the transcripts of 90 genes simultaneously on the same section of tissue from the maize (Zea mays) shoot apex. The RNA ISS technology reported expression profiles that were highly comparable with those obtained by in situ hybridizations (ISHs) and allowed the discrimination between tissue domains. Furthermore, the application of spatial transcriptomics to the shoot apex, which inherently comprised phytomers that are in gradual developmental stages, provided a spatiotemporal sequence of transcriptional events. We illustrate the power of the technology through PLASTOCHRON1 (PLA1), which was specifically expressed at the boundary between indeterminate and determinate cells and partially overlapped with ROUGH SHEATH1 and OUTER CELL LAYER4 transcripts. Also, in the inflorescence, PLA1 transcripts localized in cells subtending the lateral primordia or bordering the newly established meristematic region, suggesting a more general role of PLA1 in signaling between indeterminate and determinate cells during the formation of lateral organs. Spatial transcriptomics builds on RNA ISH, which assays relatively few transcripts at a time and provides a powerful complement to single-cell transcriptomics that inherently removes cells from their native spatial context. Further improvements in resolution and sensitivity will greatly advance research in plant developmental biology., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Muscle weakness is a frequently occurring complication of sepsis, associated with increased morbidity and mortality. Interestingly, obesity attenuates sepsis-induced muscle wasting and weakness. As the adipokine leptin is strongly elevated in obesity and has been shown to affect muscle homeostasis in non-septic conditions, we aimed to investigate whether leptin mediates the protective effect of obesity on sepsis-induced muscle weakness., Methods: In a mouse model of sepsis, we investigated the effects of genetic leptin inactivation in obese mice (leptin-deficient obese mice vs. diet-induced obese mice) and of leptin supplementation in lean mice (n = 110). We assessed impact on survival, body weight and composition, markers of muscle wasting and weakness, inflammation, and lipid metabolism. In human lean and overweight/obese intensive care unit (ICU) patients, we assessed markers of protein catabolism (n = 1388) and serum leptin (n = 150)., Results: Sepsis mortality was highest in leptin-deficient obese mice (53% vs. 23% in diet-induced obese mice and 37% in lean mice, P = 0.03). Irrespective of leptin, after 5 days of sepsis, lean mice lost double the amount of lean body mass than obese mice (P < 0.0005). Also, irrespective of leptin, obese mice maintained specific muscle force up to healthy levels (P = 0.3) whereas lean mice suffered from reduced specific muscle force (72% of healthy controls, P < 0.0002). As compared with lean septic mice, both obese septic groups had less muscle atrophy, liver amino acid catabolism, and inflammation with a 50% lower plasma TNFα increase (P < 0.005). Conversely, again mainly irrespective of leptin, obese mice lost double amount of fat mass than lean mice after 5 days of sepsis (P < 0.0001), showed signs of increased lipolysis and ketogenesis, and had higher plasma HDL and LDL lipoprotein concentrations (P ≤ 0.01 for all). Muscle fibre type composition was not altered during sepsis, but a higher atrophy sensitivity of type IIb fibres compared with IIa and IIx fibres was observed, independent of obesity or leptin. After 5 days of critical illness, serum leptin was higher (P < 0.0001) and the net waste of nitrogen (P = 0.006) and plasma urea-to-creatinine ratio (P < 0.0001) was lower in overweight/obese compared with lean ICU human patients., Conclusions: Leptin did not mediate the protective effect of obesity against sepsis-induced muscle wasting and weakness in mice. Instead, obesity-independent of leptin-attenuated inflammation, protein catabolism, and dyslipidaemia, pathways that may play a role in the observed muscle protection., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Plant organ size and shape are major agronomic traits that depend on cell division and expansion, which are both regulated by complex gene networks. In several eudicot species belonging to the rosid clade, organ growth is controlled by a repressor complex consisting of PEAPOD (PPD) and KINASE-INDUCIBLE DOMAIN INTERACTING (KIX) proteins. The role of these proteins in asterids, which together with the rosids constitute most of the core eudicot species, is unknown. We used Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 genome editing to target SlKIX8 and SlKIX9 in the asterid model species tomato (Solanum lycopersicum) and analyzed loss-of-function phenotypes. Loss-of-function of SlKIX8 and SlKIX9 led to the production of enlarged, dome-shaped leaves and these leaves exhibited increased expression of putative Solanum lycopersicum PPD (SlPPD target genes. Unexpectedly, kix8 kix9 mutants carried enlarged fruits with increased pericarp thickness due to cell expansion. At the molecular level, protein interaction assays indicated that SlKIX8 and SlKIX9 act as adaptors between the SlPPD and SlTOPLESS co-repressor proteins. Our results show that KIX8 and KIX9 are regulators of organ growth in asterids and can be used in strategies to improve important traits in produce such as thickness of the fruit flesh., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex/cyclosome (APC/C) that controls unidirectional cell cycle progression in Arabidopsis (Arabidopsis thaliana), but so far its role has not been studied in monocots. Here, we show the association of SAMBA with the APC/C is conserved in maize (Zea mays). Two samba genome edited mutants showed growth defects, such as reduced internode length, shortened upper leaves with erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion, which aggravated with plant age. The two mutants differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knockout allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity and efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: Sepsis is hallmarked by high plasma cortisol/corticosterone (CORT), low adrenocorticotropic hormone (ACTH), and high pro-opiomelanocortin (POMC). While corticotropin-releasing hormone-(CRH) and arginine-vasopressin (AVP)-driven pituitary POMC expression remains active, POMC processing into ACTH becomes impaired. Low ACTH is accompanied by loss of adrenocortical structure, although steroidogenic enzymes remain expressed. We hypothesized that treatment of sepsis with hydrocortisone (HC) aggravates this phenotype whereas CRH infusion safeguards ACTH-driven adrenocortical structure., Methods: In a fluid-resuscitated, antibiotics-treated mouse model of prolonged sepsis, we compared the effects of HC and CRH infusion with placebo on plasma ACTH, POMC, and CORT; on markers of hypothalamic CRH and AVP signaling and pituitary POMC processing; and on the adrenocortical structure and markers of steroidogenesis. In adrenal explants, we studied the steroidogenic capacity of POMC., Results: During sepsis, HC further suppressed plasma ACTH, but not POMC, predominantly by suppressing sepsis-activated CRH/AVP-signaling pathways. In contrast, in CRH-treated sepsis, plasma ACTH was normalized following restoration of pituitary POMC processing. The sepsis-induced rise in markers of adrenocortical steroidogenesis was unaltered by CRH and suppressed partially by HC, which also increased adrenal markers of inflammation. Ex vivo stimulation of adrenal explants with POMC increased CORT as effectively as an equimolar dose of ACTH., Conclusions: Treatment of sepsis with HC impaired integrity and function of the hypothalamic-pituitary-adrenal axis at the level of the pituitary and the adrenal cortex while CRH restored pituitary POMC processing without affecting the adrenal cortex. Sepsis-induced high-circulating POMC may be responsible for ongoing adrenocortical steroidogenesis despite low ACTH., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Background: In septic mice, supplementing parenteral nutrition with 150 mg/day 3-hydroxybutyrate-sodium-salt (3HB-Na) has previously shown to prevent muscle weakness without obvious toxicity. The main objective of this study was to identify the toxic threshold of 3HB-Na supplementation in septic mice, prior to translation of this promising intervention to human use., Methods: In a centrally-catheterized, antibiotic-treated, fluid-resuscitated, parenterally fed mouse model of prolonged sepsis, we compared with placebo the effects of stepwise escalating doses starting from 150 mg/day 3HB-Na on illness severity and mortality (n = 103). For 5-day survivors, also the impact on ex-vivo-measured muscle force, blood electrolytes, and markers of vital organ inflammation/damage was documented., Results: By doubling the reference dose of 150 mg/day to 300 mg/day 3HB-Na, illness severity scores doubled (p = 0.004) and mortality increased from 30.4 to 87.5 % (p = 0.002). De-escalating this dose to 225 mg still increased mortality (p ≤ 0.03) and reducing the dose to 180 mg/day still increased illness severity (p ≤ 0.04). Doses of 180 mg/day and higher caused more pronounced metabolic alkalosis and hypernatremia (p ≤ 0.04) and increased markers of kidney damage (p ≤ 0.05). Doses of 225 mg/day 3HB-Na and higher caused dehydration of brain and lungs (p ≤ 0.05) and increased markers of hippocampal neuronal damage and inflammation (p ≤ 0.02). Among survivors, 150 mg/day and 180 mg/day increased muscle force compared with placebo (p ≤ 0.05) up to healthy control levels (p ≥ 0.3)., Conclusions: This study indicates that 150 mg/day 3HB-Na supplementation prevented sepsis-induced muscle weakness in mice. However, this dose appeared maximally effective though close to the toxic threshold, possibly in part explained by excessive Na + intake with 3HB-Na. Although lower doses were not tested and thus might still hold therapeutic potential, the current results point towards a low toxic threshold for the clinical use of ketone salts in human critically ill patients. Whether 3HB-esters are equally effective and less toxic should be investigated., (© 2021. The Author(s).)

The plant hormone auxin, a master coordinator of development, regulates hypocotyl elongation during seedling growth. We previously identified the synthetic molecule RubNeddin 1 (RN1), which induces degradation of the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors INDOLE-3-ACETIC ACID-INDUCIBLE3 (IAA3) and IAA7 in planta and strongly promotes hypocotyl elongation. In the present study, we show that despite the structural similarity of RN1 to the synthetic auxin 2,4-dichlorophenoxyacetic-acid (2,4-D), direct treatments with these compounds in Arabidopsis (Arabidopsis thaliana) result in distinct effects, possibly due to enhanced uptake of RN1 and low-level, chronic release of 2,4-D from RN1 in planta. We confirm RN1-induced hypocotyl elongation occurs via specific TRANSPORT INHIBITOR RESISTANT1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) receptor-mediated auxin signaling involving TIR1, AFB2, and AFB5. Using a transcriptome profiling strategy and candidate gene approach, we identify the genes ZINC FINGER OF ARABIDOPSIS THALIANA10 (ZAT10), ARABIDOPSIS TOXICOS EN LEVADURA31 (ATL31), and WRKY DNA-BINDING PROTEIN33 (WRKY33) as being rapidly upregulated by RN1, despite being downregulated by 2,4-D treatment. RN1-induced expression of these genes also occurs via TIR1/AFB-mediated auxin signaling. Our results suggest both hypocotyl elongation and transcription of these genes are induced by RN1 via the promoted degradation of the AUX/IAA transcriptional repressor IAA7. Moreover, these three genes, which are known to be stress-related, act in an inter-dependent transcriptional regulatory network controlling hypocotyl elongation. Together, our results suggest ZAT10, ATL31, and WRKY33 take part in a common gene network regulating hypocotyl elongation in Arabidopsis downstream of a selective auxin perception module likely involving TIR1, AFB2, and AFB5 and inducing the degradation of IAA7., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: To investigate the effect of Neuromuscular Electrical Stimulation (NMES) on muscle thickness, strength and morphological and molecular markers of the quadriceps., Materials and Methods: Adult critically ill patients with an expected prolonged stay received unilateral quadriceps NMES sessions for 7 consecutive days. Before and after the intervention period, quadriceps thickness was measured with ultrasound. After the intervention period, strength was assessed in cooperative patients and muscle biopsies were taken. Multivariable regression was performed to identify factors affecting muscle thickness loss., Results: Muscle thickness decreased less in the stimulated leg (-6 ± 16% versus -12 ± 15%, p = 0.014, n = 47). Strength was comparable. Opioid administration, minimal muscle contraction and more muscle thickness loss in the non-stimulated muscle were independently associated with better muscle thickness preservation. Stimulated muscles showed a shift towards larger myofibers and higher MyHC-I gene expression. NMES did not affect gene expression of other myofibrillary proteins, MuRF-1 or atrogin-1. Signs of myofiber necrosis and inflammation were comparable for both muscles., Conclusions: NMES attenuated the loss of muscle mass, but not of strength, in critically ill patients. Preservation of muscle mass was more likely in patients receiving opioids, patients with a minimal muscle contraction during NMES and patients more prone to lose muscle mass., Trial Registration: clinicaltrials.govNCT02133300., Competing Interests: Declaration of Competing Interest None of the authors have declared any conflict of interest related to the subject of this study. The authors acknowledge DJO Global (Herentals, Belgium) for providing the neuromuscular stimulation equipment used in this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)