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The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments. Combinatorial Gene×Gene×Environment CRISPR screen targeting human SLC25 transporter family enables the identification of SLC25A39 in mitochondrial glutathione import and its coordination with mitochondrial iron import in supporting OXPHOS.

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

Background Pulmonary infection is common yet serious complication in patients with severe traumatic brain injury (STBI). We aimed to evaluate the predicators of pulmonary infection in STBI patients undergoing tracheostomy, to provide evidence for the clinical nursing care of STBI patients. Methods This study was a retrospective cohort design. STBI patients undergoing tracheostomy treatment from January 1, 2019 to August 31, 2021 in our hospital were included. The characteristics of pulmonary infection and no pulmonary infection patients were analyzed. Results A total 216 STBI patients undergoing tracheostomy were included, the incidence of pulmonary infection was 26.85%. Diabetes (r = 0.782), hypoproteinemia (r = 0.804), duration of coma(r = 0.672), duration of mechanical ventilation(r = 0.724) and length of hospital stay (r = 0.655), length of hospital stay post tracheostomy (r = 0.554), mortality (r = 0.598) were all correlated with pulmonary infection (all p  < 0.05). Klebsiella pneumoniae (33.87%) and Staphylococcus aureus (29.03%) were the most commonly seen pathogens in the pulmonary infection of TBI patients. Logistic regression analyses indicated that diabetes (OR 2.232, 95% CI 1.215–3.904), hypoproteinemia with plasma total protein < 60 g/L (OR 1.922, 95% CI 1.083–3.031), duration of coma ≥ 22 h (OR 2.864, 95% CI 1.344–5.012), duration of mechanical ventilation ≥ 5 days (OR 3.602, 95% CI 1.297–5.626), length of hospital stay ≥ 21 days (OR 2.048, 95% CI 1.022–3.859) were the risk factors of pulmonary infection in TBI patients undergoing tracheostomy (all p  < 0.05). Conclusions Further investigations on the early preventions and treatments targeted on those risk factors are needed to reduce the pulmonary infection in clinical practice.

Hirudo nipponia is an important medicinal animal in China. Its salivary gland secretions contain a variety of protein bioactive substances. Investigations of its salivary glands are of great significance in the study of the medicinal value and mechanism of leech secretions. Illumina RNA-Seq technology was used to perform transcriptome sequencing of salivary gland tissue of H . nipponia under starvation (D30) and fed (D0) states. A total of 2,650 differentially expressed genes (DEGs) were screened. Using the label-free protein quantification technique and bioinformatics analysis, the expression of differentially expressed proteins (DEPs) in the salivary gland tissue of H . nipponia was compared. A total of 2,021 proteins were identified, among which 181 proteins were differentially expressed between the starvation and fed states, with 72 significantly upregulated and 109 significantly downregulated. The salivary glands of H . nipponia synthesized protein-based active substances after 30 days of starvation and adapted to the starvation environment by weakening respiratory activity and reducing metabolic activity to reduce energy expenditure. Energy was produced by glycolysis and the tricarboxylic acid cycle for the synthesis of substances such as antibiotics. This study combined transcriptome and proteome sequencing data to provide a data reference for an in-depth study of the regulatory mechanism of salivary gland secretions of H . nipponia under starvation stress by analyzing DEGs and DEPs.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1 -KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD. Mutations in the gene encoding PC1 cause ADPKD, a common genetic renal disease. Here, the authors show that expression of the C-terminal 200 amino acids of the large PC1 protein in mouse models of ADPKD suppresses cystic disease through an interaction with the mitochondrial enzyme NNT.

Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production. Despite this utility, broad deployment has been hindered by technical limitations and an inability to reconstruct complex phopho-regulatory networks. Here, we address these challenges by optimizing genetically encoded phosphothreonine translation to characterize phospho-dependent kinase activation mechanisms and, subsequently, develop a multi-level protein interaction platform to directly assess the overlap of kinase and phospho-binding protein substrate networks with phosphosite-level resolution. Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. Here the authors optimize genetically encoded phosphothreonine to study the regulation of CHK2 kinase using large-scale DNA arrays that enable phosphoproteome expression techniques to identify sitespecific overlap between CHK2 substrates and 14-3-3 interactions.

The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.

This study addresses the issue of poor construction labor productivity, exploring the process and methodology for increasing construction workers’ participation in labor productivity improvement, as well as a situational awareness (SA) development approach for construction workers based on lean construction (LC). A framework for enhancing labor productivity through SA has been developed. A case study approach is utilized to describe the LC improvement process, which includes enhancing construction workers’ SA for waste minimization and process optimization and engaging them in LC with improved value stream mapping (VSM). The labor productivity measurement approach, combined with VSM, is used to evaluate the labor productivity improvement with labor productivity improvement rate, resource efficiency, and flow efficiency. The results demonstrate that the labor productivity of the case project has increased by 24.07%, with 11.54% contributing resource efficiency and 88.46% contributing flow efficiency, indicating a greater increase in flow efficiency. This study fills a research gap in terms of the combination of construction workers’ SA enhancement and the involvement of LC practices to boost labor productivity. Furthermore, this study proposes an SA development approach based on LC at three levels: waste perception, VSM implementation, and labor productivity improvement assessment. This provides new perspectives for leveraging construction workers’ SA to increase labor productivity. The improved VSM approach and labor productivity measurements provide an operationalization of workers’ participation in continuous improvement. This research is of great practical relevance for augmenting the competencies of construction laborers and inspiring them to engage in labor productivity enhancement.

Glucose is vital for life, serving as both a source of energy and carbon building block for growth. When glucose is limiting, alternative nutrients must be harnessed. To identify mechanisms by which cells can tolerate complete loss of glucose, we performed nutrient-sensitized genome-wide genetic screens and a PRISM growth assay across 482 cancer cell lines. We report that catabolism of uridine from the medium enables the growth of cells in the complete absence of glucose. While previous studies have shown that uridine can be salvaged to support pyrimidine synthesis in the setting of mitochondrial oxidative phosphorylation deficiency 1 , our work demonstrates that the ribose moiety of uridine or RNA can be salvaged to fulfil energy requirements via a pathway based on: (1) the phosphorylytic cleavage of uridine by uridine phosphorylase UPP1/UPP2 into uracil and ribose-1-phosphate (R1P), (2) the conversion of uridine-derived R1P into fructose-6-P and glyceraldehyde-3-P by the non-oxidative branch of the pentose phosphate pathway and (3) their glycolytic utilization to fuel ATP production, biosynthesis and gluconeogenesis. Capacity for glycolysis from uridine-derived ribose appears widespread, and we confirm its activity in cancer lineages, primary macrophages and mice in vivo. An interesting property of this pathway is that R1P enters downstream of the initial, highly regulated steps of glucose transport and upper glycolysis. We anticipate that ‘uridine bypass’ of upper glycolysis could be important in the context of disease and even exploited for therapeutic purposes. In this study, Skinner, Blanco-Fernández et al. show that uridine can be salvaged through the non-oxidative branch of the pentose phosphate pathway to feed glycolysis in conditions of glucose scarcity.

To investigate the effect of feeding on hibernating Hirudo nipponia, metagenomic sequencing was performed on the microorganisms collected from the digestive tract of H. nipponia individuals that were fed (FW) or starving (SW) before hibernation. In addition, the salivary gland tissues were obtained from these individuals to conduct transcriptome sequencing. Results showed that feeding before hibernation increased the relative abundance of Bacteroidetes and Proteobacteria at the phylum level and that of Aeromonas, Mucinivorans, Bacteroides, and Desulfovibrio at the genus level. Functional prediction results indicated that feeding before hibernation not only improved metabolic capacity but also increased the susceptibility of H. nipponia to pathogens. Among the 133,082 unigenes obtained by transcriptome sequencing, a total of 2801 differentially expressed genes (DEGs) were found, of which 355 were up-regulated and 2446 were down-regulated; the expression patterns of 15 selected DEGs were validated by qRT-PCR. Results from the GO and KEGG enrichment analyses showed that a majority of these DEGs were involved in the signal transduction pathways. This study established a basis for H. nipponia breeding during hibernation through analyses of the abundance of microorganisms in the digestive tract and the gene expression pattern of salivary glands by high-throughput sequencing. The findings also revealed the effect of feeding on the expression of both gastrointestinal microorganisms and salivary gland genes of hibernating H. nipponia.