Biology subjects

Greene, N. P.

Publications and source records attributed to Greene, N. P..

7 recordsLinked to original sources

bioRxiv2026

Structures of LolB bound to LolA or lipoprotein resolve the final steps of bacterial lipoprotein trafficking

In Gram-negative bacteria, lipoproteins are structural elements of the outer membrane and essential components of machineries responsible for its construction and maintenance. The Lol system, responsible for the trafficking of lipoproteins from the site of maturation on the inner membrane to the outer membrane, is therefore crucial to the function of the cell envelope and a key target of efforts to find novel antimicrobials. In the final steps of this process, the outer membrane receptor, LolB accepts triacylated lipoproteins from the periplasmic chaperone LolA before inserting them into the outer membrane. Here we present a structure of LolB in complex with LolA, validated by in vivo and in vitro assays, highlighting how positively charged residues on the convex face of the LolB {beta}-barrel underpin complex formation. A protruding loop of LolB, essential for function, inserts into the LolA cavity in position to initiate the displacement of substrate lipoprotein from LolA to enable transfer to LolB. Structural resolution of a lipoprotein-bound LolB complex in combination with biophysical assays shows how a molecular latch releases the lid of the cavity to accommodate the lipoprotein acyl chains. Modelling of these structures onto computationally predicted orientations for LolB on the outer membrane provides a rationale for LolA release and lipoprotein triacyl group membrane insertion. Taken altogether, our data elucidate atomic resolution of two key intermediates and provide a greater understanding of the terminal steps of lipoprotein trafficking events at the bacterial outer membrane.

microbiology↗

bioRxiv2025

The Age-Dependent Resident Myonuclear Multi-Omic Response to a Skeletal Muscle Hypertrophic Stimulus

A detailed analysis of how muscle fiber nuclei (myonuclei) respond to a hypertrophic stimulus would provide a critical step toward understanding compromised skeletal muscle plasticity with age. We used recombination-independent doxycycline-inducible myonucleus-specific fluorescent labelling, tissue RNA-sequencing, myonuclear DNA methylation analysis, multi-omic integration, and single myonucleus RNA-sequencing to define the molecular characteristics of adult (6-8 month) and aged (24 month) murine skeletal muscle after acute mechanical overload (MOV). In adult and aged MOV muscles, we found that: 1) similarities in the transcriptional response to loading - specifically in metabolism genes - were partly explained by a post-transcriptional microRNA-mediated mechanism, which we corroborated using an inducible muscle fiber-specific miR-1 knockout model, 2) differences in age-dependent transcriptional responses were linked to the magnitude and location of differential DNA methylation in resident myonuclei, specifically around hypertrophy-associated genes such as Myc, Runx1, Mybph, Ankrd1, collagen genes, and minichromosome maintenance genes, 3) adult and aged resident myonuclear transcriptomes had differing enrichment for innervation-related transcripts as well as unique transcriptional profiles in an Atf3+ "sarcomere assembly" population after MOV, and 4) cellular deconvolution analysis supports a role for neuromuscular junction regulation in age-specific hypertrophic adaptation. These data are a roadmap for uncovering molecular targets to enhance aged muscle adaptability.

cell biology↗

bioRxiv2024

Mitochondrial-targeted plastoquinone therapy ameliorates early onset muscle weakness that precedes ovarian cancer cachexia in mice

Cancer cachexia, and the related loss of muscle and strength, worsens quality of life and lowers overall survival. Recently, a novel pre-atrophy muscle weakness was identified during early-stage cancer. While mitochondrial stress responses are associated with early-stage pre-atrophy weakness, a causal relationship has not been established. Using a robust mouse model of metastatic epithelial ovarian cancer (EOC)-induced cachexia, we found the well-established mitochondrial-targeted plastoquinone SkQ1 partially prevents pre-atrophy weakness in the diaphragm. Furthermore, SkQ1 improved force production during atrophy without preventing atrophy itself in the tibialis anterior and diaphragm. EOC reduced flexor digitorum brevis (FDB) force production and myoplasmic free calcium ([Ca2+]i) during contraction in single muscle fibers, both of which were prevented by SkQ1. Remarkably, changes in mitochondrial reactive oxygen species and pyruvate metabolism were heterogeneous across time and between muscle types which highlights a considerable complexity in the relationships between mitochondria and muscle remodeling throughout EOC. These discoveries identify that muscle weakness can occur independent of atrophy throughout EOC in a manner that is linked to improved calcium handling. The findings also demonstrate that mitochondrial-targeted therapies exert a robust effect in preserving muscle force during the early pre-atrophy period and in late-stage EOC once cachexia has become severe.

physiology↗

bioRxiv2024

Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia

ObjectivesA high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. MethodsHere, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm [~]45, [~]75 and [~]90 days after EOC injection. ResultsPrimary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNF), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. ConclusionThis mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia. HighlightsO_LIThis study reports the first orthotopic model of metastatic ovarian cancer cachexia that can be induced in adult immunocompetent mice C_LIO_LIDiaphragm and limb muscle weakness precedes metastasis and atrophy during ovarian cancer C_LIO_LISkeletal muscle mitochondrial oxidative and redox stress signatures occur during pre-metastatic stages of ovarian cancer C_LIO_LISpecific muscle force as well as mitochondrial pyruvate oxidation and creatine metabolism demonstrate compensation in later stages C_LIO_LIOvarian cancer has heterogeneous effects on distinct muscle types across time C_LI

physiology↗

bioRxiv2024

The 24-Hour Time Course of Integrated Molecular Responses to Resistance Exercise in Human Skeletal Muscle Implicates MYC as a Hypertrophic Regulator That is Sufficient for Growth

Molecular control of recovery after exercise in muscle is temporally dynamic. A time course of biopsies around resistance exercise (RE) combined with -omics is necessary to better comprehend the molecular contributions of skeletal muscle adaptation in humans. Vastus lateralis biopsies before and 30 minutes, 3-, 8-, and 24-hours after acute RE were collected. A time-point matched biopsy-only group was also included. RNA-sequencing defined the transcriptome while DNA methylomics and computational approaches complemented these data. The post-RE time course revealed: 1) DNA methylome responses at 30 minutes corresponded to upregulated genes at 3 hours, 2) a burst of translation- and transcription-initiation factor-coding transcripts occurred between 3 and 8 hours, 3) global gene expression peaked at 8 hours, 4) ribosome-related genes dominated the mRNA landscape between 8 and 24 hours, 5) methylation-regulated MYC was a highly influential transcription factor throughout the 24-hour recovery and played a primary role in ribosome-related mRNA levels between 8 and 24 hours. The influence of MYC in human muscle adaptation was strengthened by transcriptome information from acute MYC overexpression in mouse muscle. To test whether MYC was sufficient for hypertrophy, we generated a muscle fiber-specific doxycycline inducible model of pulsatile MYC induction. Periodic 48-hour pulses of MYC over 4 weeks resulted in higher muscle mass and fiber size in the soleus of adult female mice. Collectively, we present a temporally resolved resource for understanding molecular adaptations to RE in muscle and reveal MYC as a regulator of RE-induced mRNA levels and hypertrophy.

cell biology↗

bioRxiv2022

The Time-Course of Cancer Cachexia Onset Reveals Biphasic Transcriptional Disruptions in Female Skeletal Muscle Distinct from Males

BackgroundCancer-cachexia (CC) is experienced by 80% of cancer patients, representing 40% of cancer-related deaths. Evidence suggests biological sex dimorphism is associated with CC. Assessments of the female transcriptome in CC are lacking and direct comparisons between biological sex are scarce. The purpose of this study was to define the time course of LLC-induced CC in females using transcriptomics, while directly comparing the effects of biological sex. MethodsEight-week-old female mice were injected with LLC cells (1x106) or sterile PBS to the hind flank. Tumors developed for 1, 2, 3 or 4-weeks. Due to dimorphism between tumor weight in 3- and 4-weeks of development, these were reorganized as low-tumor weight (LT, tumor-weight [≤]1.2g), or high-tumor weight (HT, tumor-weight [≥]2g). Gastrocnemius muscle was collected for RNA-sequencing (RNA-seq). Differentially expressed genes (DEGs) were defined as FDR<0.05. Data were further compared to RNA-seq of male mice from a previous study. ResultsGlobal gene expression of female gastrocnemius muscle reveals consistent DEGs at all timepoints, all associated with type-II interferon signaling (FDR<0.05). Early transcriptomic upregulation of extracellular-matrix pathways was noted at 1wk (p<0.05), JAK-STAT pathway was upregulated in 2wk, LT, and HT. Type II interferon signaling was downregulated in 1wk, LT, and HT (p<0.05). A second major transcriptomic downregulation in oxidative phosphorylation, electron transport chain and TCA cycle were noted in cachectic (HT) muscle only (p<0.05). Male-female comparison of cachectic groups revealed 69% of DEGs were distinct between sex (FDR<0.05). Comparison of the top 10-up and down DEGs revealed downregulation of type-II Interferon genes was unique to female, while males show upregulation of interferon-signaling pathways. ConclusionWe demonstrate biphasic disruptions in transcriptome of female LLC tumor-bearing mice: an early phase associated with ECM remodeling and a late phase, accompanied by onset of systemic cachexia, affecting overall skeletal muscle energy metabolism. Comparison of cachectic female-male mice reveals ~2/3 of DEGs are biological sex specific, providing evidence of dimorphic mechanisms of cachexia between sexes. Alterations to Type-II Interferon signaling appears specific to CC development in females, suggesting a new biological sex-specific marker of CC. Our data support biological sex dimorphisms in development of CC. HighlightsO_LIWhile males show impairments in skeletal muscle energy metabolism in early stages of CC, early transcriptomic alterations impact ECM remodeling that precedes impairments in skeletal muscle energy metabolism in female tumor-bearing mice. C_LIO_LI2/3 of differently expressed genes in skeletal muscle undergoing cachexia are biological sex specific. C_LIO_LIDownregulation of Type-II Interferon genes is unique to female mice, which displayed preserved gastrocnemius mass despite systemic cachexia, representing a potential therapeutic target for muscle mass maintenance in cancer-induced atrophy. C_LIO_LIMechanisms of LLC-induced cachexia appear to be biological sex specific which needs to be considered in further study of mechanisms and therapeutic modalities. C_LI

bioinformatics↗

bioRxiv2021

Muscle weakness precedes atrophy during cancer cachexia and is associated with muscle-specific mitochondrial stress

Muscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but distinct responses between muscles and across time remains unclear. We aimed to determine the time-dependent and muscle-specific responses to Colon-26 (C26) cancer-induced cachexia in mice. At 2 weeks post-inoculation, the presence of small tumours did not alter body or muscle mass but decreased force production in the quadriceps and diaphragm. Pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H2O2 emission was elevated in diaphragm. At 4 weeks, large tumours corresponded to lower body mass, muscle mass, and cross-sectional area of fibers in quadriceps and diaphragm. Force production in quadriceps was unchanged but remained lower in diaphragm vs control. Mitochondrial respiration was increased while H2O2 emission was unchanged in both muscles vs control. Mitochondrial creatine sensitivity was compromised in quadriceps. These findings indicate muscle weakness precedes atrophy in quadriceps and diaphragm but is linked to heterogeneous mitochondrial alterations. Eventual muscle-specific restorations in force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscles to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.

physiology↗