Biology subjects

Wallace, M.

Publications and source records attributed to Wallace, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Listianol, a Carotenoid Biosynthesis Inhibitor from a Methylotrophic Bacterium, Alters the Composition of a Phyllosphere Microbial Community

The microbial communities that inhabit the phyllosphere, the above-ground portion of plants, are important for plant growth and resilience. However, the natural products that mediate interactions among these organisms and with their environment remain understudied, limiting our molecular-level understanding of how phyllosphere microbial communities are structured and maintained. Natural product-mediated microbial competition is typically associated with growth inhibition, but may also occur through other mechanisms in these communities. Many bacteria produce pigments to mitigate oxidative stress, and here we identify listianol, a previously undescribed natural product that inhibits the pigmentation of diverse bacteria. Listianol blocks carotenoid biosynthesis by targeting the desaturases CrtN and CrtI. This activity sensitizes normally pigmented bacteria to UVB radiation in vitro, and a listianol-producing strain reshapes the composition of a model bacterial community on Arabidopsis thaliana under UVB exposure. Together, these findings identify pigmentation inhibition as a potential form of competition in the phyllosphere.

microbiology↗

bioRxiv2026

An AI/ML-Powered Workflow for End-to-End Cell Line Development

The generation of clonal CHO cell lines is foundational to biologics manufacturing; however, labor-intensive cell culture workflows predominate in the field. We created the CLAIRE (Cell Line AI Recognition and Evaluation) tool to streamline end-to-end cell line development by integrating deep-learning image analysis with automated liquid handling. We benchmarked three object detection models for monoclonality verification and found DETR provides superior accuracy (>0.90 F1-score) in identifying single cells. To quantify the outgrowth of cell lines, we evaluated multiple zero-shot SAM2 segmentation models against a feature-based estimation method. Feature-based detection successfully identified diverse cell colony types while less robust performance was observed for SAM2 models, particularly for sparse density colonies. The pre-trained DETR and feature-based detection models were wrapped in a task-focused user interface that outputs cell line hitpick lists compatible with a Lynx LM1800 liquid handler in addition to custom scripts automating cell passaging and sampling. This approach yielded an end-to-end 36 day CLD workflow capable of generating high-titer cell lines for multiple complex antibody structures. Here, we open-access our trained models, user interface, and Lynx automation scripts to provide a modular toolkit useful for clonal cell line engineering projects. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/703387v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f72e70org.highwire.dtl.DTLVardef@109c54dorg.highwire.dtl.DTLVardef@7867b1org.highwire.dtl.DTLVardef@dfa61e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Enhanced Zika Virus Suppression by Coupling Ribavirin with Biodegradable Drug Delivery

Advancement and implementation of nanoparticle-based systems for enhanced drug delivery have become essential for the suppression of viral pathogens due to lack of viable treatment options. This study focuses on characterization and application of mPEG-PCL co-polymers for development of pH-responsive, biodegradable micellar nanoparticles (MNPs) for the delivery of the antiviral drug ribavirin against Zika virus. Synthesis of mPEG-PCL diblock copolymers was confirmed through FTIR and 1H NMR, which validated the formation of ester functional groups and accurate structure of copolymers. mPEG-PCL MNPs, loaded with ribavirin, exhibited a mean diameter of approximately 34.29 {+/-} 5.214 nm and a zeta potential of -3.28 {+/-} 0.718 mV, suitable for evading immune detection and facilitating cellular entry. The ribavirin-loaded mPEG-PCL MNPs demonstrated pH-dependent release, with approximately 88% of ribavirin released at pH 5.5, compared to 20% at pH 7.4. This pH-responsive release is crucial for targeted drug delivery within the endosomal pathway. In vitro studies using JEG-3 cells infected with Zika virus showed that ribavirin-loaded mPEG-PCL MNPs achieved an EC50 of 0.22nM, significantly enhancing drug efficacy compared to unencapsulated ribavirin, which required micromolar concentrations to achieve similar effects. The MTT assay results indicated minimal cytotoxicity of the ribavirin-loaded mPEG-PCL MNPs, with approximately 80% cell viability at the highest concentration evaluated. Confocal microscopy and RT-PCR analysis further confirmed the efficient cellular uptake and potent antiviral activity of the ribavirin-loaded MNPs. These findings highlight the potential of mPEG-PCL MNPs as an effective delivery system for broad-spectrum antivirals like ribavirin, enhancing their therapeutic efficacy while minimizing cytotoxicity.

microbiology↗

bioRxiv2025

Dynamic exchange of antimicrobial peptides stabilize persistent lipid pores

Antimicrobial peptides (AMPs) are a promising broad-spectrum complement to conventional antibiotics. But despite their relatively simple structures, the reported modes of action of AMPs are diverse, ranging from formation of stable ion-channel-like structures, or disruption of biochemical pathways, to comprehensive membrane solubilization. This apparent complexity of function is a significant hurdle in their future application and rational design. Here, we combine single-molecule tracking with optical single-channel recording in synthetic mimics of bacterial membranes to image the pores formed by AMPs in real time, mapping the diffusion of individual AMPs relative to sites of membrane permeablization. Using alamethicin, magainin-II and melittin as archetypes of three principal membrane-disrupting AMP classes, we observe AMP molecules freely diffusing on the membrane, but with an enhancement of local peptide surface density within a nanoscopic region surrounding each pore locus. We do not observe this enhancement for indolicidin, an AMP which is not believed to use pore formation as its main mode of toxicity. Corroborated by molecular dynamics simulations that replicate our experiments, these results suggest defects formed by membrane-active AMPs are persistent but dynamic structures, stabilised by individual peptides free to diffuse into and out of the pore. Whilst alamethicin, magainin-II and melittin are often placed in different mechanistic pore-forming groups, the broad similarities between observations in these different AMPs encourages a continuum view of pore-forming ability, rather than discrete categories of mechanism.

biophysics↗

bioRxiv2025

TBK1 activity regulates the directionality of axonal transport of signalling endosomes

The polarised and complex morphology of neurons pose massive challenges for efficient cargo delivery between the axon and soma, a process termed axonal transport. We have previously shown that the retrograde axonal transport of pro-survival, neurotrophic signalling endosomes relies on Rab7 in motor neurons, and that their trafficking is impaired in the early stages of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we report the effect of Rab7 phosphorylation on the transport of these signalling endosomes. We show that the ALS-linked kinase TBK1 phosphorylates Rab7 at S72 in neurons, altering its binding to cytoplasmic dynein adaptors. Accordingly, both TBK1 knockdown and the expression of a loss-of-function Rab7 mutant (S72E) induce aberrant bidirectional movement of signalling endosomes without modifying neuronal polarity or endosomal sorting. This alteration is specific for signalling endosomes, as axonal transport of lysosomes and mitochondria remain unaffected. We have therefore discovered a new TBK1 function that ensures the unidirectional transport of signalling endosomes, suggesting that reduced TBK1 activity determines retrograde transport dysfunctions and long-range signalling impairments. SummarySignalling endosomes travel from the distal axon to the cell body, exerting pro-survival functions. TBK1, an ALS-linked kinase, governs the directionality of signalling endosome transport in motor neurons by phosphorylating Rab7. Thus, loss of TBK1 function results in long-range signalling impairment.

neuroscience↗

bioRxiv2025

Integration of Audiovisual Motion in Dorsolateral Prefrontal Cortical Neurons

The dorsolateral prefrontal cortex is well recognized for its role in cognitive functions and activating action plans. In contrast, the properties of prefrontal neurons with respect to multisensory processing are less well studied. To address this question, we recorded single units from areas 8a and 46 of two female rhesus macaques while they were presented with visual, auditory, and audiovisual motion stimuli. The majority of dorsolateral prefrontal neurons responded to these sensory stimuli, with similar percentages of auditory-only, visual-only and audiovisual neurons. Approximately one third of responsive neurons exhibited significant super- or sub-additive interactions in response to the pairing of auditory and visual stimuli, revealing significant nonlinearities in their response profiles. Decoding motion signals from the population activity robustly differentiated multisensory from unisensory trials and also unisensory auditory and visual trials from each other. These results demonstrate that dorsolateral prefrontal neurons integrate auditory and visual motion signals, extending multisensory computations beyond sensory cortices into prefrontal circuits that support higher-order cognition. New & NoteworthyWe recorded single neurons in macaque dorsolateral prefrontal cortex during visual, auditory, and audiovisual motion. Nearly half of responsive neurons were multisensory and a third displayed significant super- or sub-additive interactions, while ensemble activity reliably decoded stimulus modality. These findings provide the strongest evidence to date that DLPFC performs rapid, nonlinear audiovisual integration, extending multisensory computations beyond classical posterior regions into the prefrontal circuits that support cognition.

neuroscience↗

bioRxiv2025

On-cell Saturation Transfer Difference (STD) NMR on ion channels: characterizing negative allosteric modulator binding interactions of P2X7.

P2X7 receptors are important drug targets involved in pathologies ranging from psychiatric disorders to cancer. Being membrane embedded receptors, they are challenging for structural characterisation and at present we only have a small number of X-ray and cryoEM structures for P2X7 bound to antagonists. We demonstrate that Saturation Transfer Difference (STD) NMR on live mammalian cells (on-cell STD NMR) overexpressing P2X7 receptors allows further structural insight on the complexes of P2X7 with two potent negative allosteric modulators, namely AZ10606120 and JNJ-47465567, via the determination of the binding epitope mapping of the interactions e.g. the main region of contact between the ligand and the binding pocket. This approach, reported for the first time on membrane-embedded ion channels, in combination with molecular docking, allows us to propose the first NMR-validated 3D molecular models for two antagonists as bound to human P2X7 receptors, and to correlate the structural knowledge acquired with the pharmacology data. We highlight the transformative potential of this application to aid drug design efforts in a less resource-demanding fashion than X-ray crystallography and cryo-EM and we envisage on-cell STD NMR to fast become an asset for structure-activity-relationship studies helping knowledge-based development of efficient drugs targeting P2X7 and other ion channels/membrane-embedded proteins.

pharmacology and toxicology↗

bioRxiv2024

Efficient creation and characterization of multi-kilobase knockins by CRISPR/Cas9 and multiple rAAVs

Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex (RNP) enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.6 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared to other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of the KI allele, genotypes non-KI alleles and more importantly, uniquely identifies donor concatenation in the KI allele, and localizes random integration of the full or partial donor. Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/625457v3_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1619b77org.highwire.dtl.DTLVardef@125b0b2org.highwire.dtl.DTLVardef@173b12dorg.highwire.dtl.DTLVardef@7519b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

'Disease-smart' assisted migration can enhance population fitness and increase resistance to pathogens via immune priming

O_LIWe studied the potential of combining insect immune priming to synergize with introduction of diverse migrant to safeguard small populations from disease outbreaks that might otherwise lead to extinction. C_LIO_LIImmune priming in insects refers to the stronger immune response insects have against pathogens after prior exposure. This enhanced immunity can be passed on to offspring and holds promise for insect conservation efforts against diseases. C_LIO_LIWe compared the fitness benefits to a small, inbred population of adding migrants that had not been primed to adding immune primed migrants. While both types of migrants enhanced reproduction, as in cases of genetic rescue, only primed migrants improved survival on exposure to a pathogen. C_LIO_LIBetter immunity led to a trade-off with reproduction in the migrants, but not upon outcrossing with the target population, revealing synergies between hybrid vigor and immune priming. C_LIO_LIGiven the demographic constraints and stochasticity that can exacerbate the effects of disease outbreaks in small populations, combining immune priming with assisted migration offers a proactive strategy to mitigate disease impacts. C_LI

ecology↗

bioRxiv2024

Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids

Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach strengthens our ability to quantify and characterize the inherent diversity of acyl chains across the lipidome.

biochemistry↗

bioRxiv2024

Sex-dependent adipose glucose partitioning by the mitochondrial pyruvate carrier

ObjectiveThe mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice. MethodsThe impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1AD-/-) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics. ResultsTreatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1AD-/- mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1AD-/- mice regardless of sex, even under conditions of zero dietary fat. ConclusionThese findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.

physiology↗

bioRxiv2023

Long chain monomethyl branched-chain fatty acid levels in human milk vary with gestational weight gain

Breastfeeding is an important determinant of infant health and there is immense interest in understanding its metabolite composition so that key beneficial components can be identified. The aim of this research was to measure the fatty acid composition of human milk in an Irish cohort where we examined changes depending on lactation stage and gestational weight gain trajectory. Utilising a chromatography approach optimal for isomer separation, we identified 44 individual fatty acid species via GCMS and showed that monomethyl branched-chain fatty acids(mmBCFAs), C15:0 and C16:1 are lower in women with excess gestational weight gain versus low gestational weight gain. To further explore the potential contribution of the activity of endogenous metabolic pathways to levels of these fatty acids in milk, we administered D2O to C57BL/6J dams fed a purified lard based high fat diet (HFD) or low-fat diet during gestation and quantified the total and de novo synthesized levels of fatty acids in their milk. We found that de novo synthesis over three days can account for between 10 and 50% of mmBCFAs in milk from dams on the low-fat diet dependent on the branched-chain fatty acid species. However, HFD fed mice had significantly decreased de novo synthesized fatty acids in milk resulting in lower total mmBCFAs and medium chain fatty acid levels. Overall, our findings highlight the diverse fatty acid composition of human milk and that human milk mmBCFA levels differ between gestational weight gain phenotypes. In addition, our data indicates that de novo synthesis contributes to mmBCFA levels in mice milk and thus may also be a contributory factor to mmBCFA levels in human milk. Given emerging data indicating mmBCFAs may be beneficial components of milk, this study contributes to our knowledge around the phenotypic factors that may impact their levels.

physiology↗

bioRxiv2023

The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis

Elevated levels of branched chain amino acids (BCAAs) and branched-chain -ketoacids (BCKAs) are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and BCKA levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly five-fold less potent than the prototypical uncoupler 2,4-dinitrophenol (DNP), and phenocopies DNP in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

biochemistry↗

bioRxiv2023

Near-field potentials index local neural computations more accurately than population spiking

Local field potentials (LFP) are low-frequency extracellular voltage fluctuations thought to primarily arise from synaptic activity. However, unlike highly localized neuronal spiking, LFP is spatially less specific. LFP measured at one location is not entirely generated there due to far-field contributions that are passively conducted across volumes of neural tissue. We sought to quantify how much information within the locally generated, near-field low-frequency activity (nfLFP) is masked by volume-conducted far-field signals. To do so, we measured laminar neural activity in primary visual cortex (V1) of monkeys viewing sequences of multifeatured stimuli. We compared information content of regular LFP and nfLFP that was mathematically stripped of volume-conducted far-field contributions. Information content was estimated by decoding stimulus properties from neural responses via spatiotemporal multivariate pattern analysis. Volume-conducted information differed from locally generated information in two important ways: (1) for stimulus features relevant to V1 processing (orientation and eye-of-origin), nfLFP contained more information. (2) in contrast, the volume-conducted signal was more informative regarding temporal context (relative stimulus position in a sequence), a signal likely to be coming from elsewhere. Moreover, LFP and nfLFP differed both spectrally as well as spatially, urging caution regarding the interpretations of individual frequency bands and/or laminar patterns of LFP. Most importantly, we found that population spiking of local neurons was less informative than either the LFP or nfLFP, with nfLFP containing most of the relevant information regarding local stimulus processing. These findings suggest that the optimal way to read out local computational processing from neural activity is to decode the local contributions to LFP, with significant information loss hampering both regular LFP and local spiking. Authors ContributionsConceptualization, D.A.T., J.A.W, and A.M.; Data Collection, J.A.W., M.A.C., K.D.; Formal Analysis, D.A.T. and J.A.W.; Data Visualization, D.A.T. and J.A.W.; Original Draft, D.A.T., J.A.W., and A.M.; Revisions and Final Draft, D.A.T., J.A.W., M.A.C., K.D., M.T.W., A.M.B., and A.M. Competing InterestsThe authors declare no conflicts of interest.

neuroscience↗

bioRxiv2022

The Lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope accessibility

Lamins A/C are nuclear intermediate filament proteins that are involved in diverse cellular mechanical and biochemical functions. Here, we report that recognition of Lamins A/C by a commonly used antibody (JOL-2) that binds the Lamin A/C Ig-fold and other antibodies targeting similar epitopes is highly dependent on cell density, even though Lamin A/C protein levels do not change with cell density. The density-dependent Lamin A/C labeling was distinct from previously reported differential apico-basal labeling, which was independent of cell density. Comparison of the density-dependent labeling effects of antibodies recognizing different Lamin A/C epitopes suggests that the effect is caused by partial unfolding or masking of the CE and/or EF loops of the Ig-fold in response to cell spreading. Seeding cells on micropatterned surfaces with different areas confirmed that increased cell spreading resulted in reduced Lamin A/C labeling with the JOL-2 antibody. Surprisingly, JOL-2 antibody labeling was insensitive to depolymerization of cytoskeletal filaments or disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Although the density-dependent changes of the Ig-fold did not alter nuclear stiffness or nucleo-cytoskeletal force transmission, they may nonetheless modulate interaction with lamin binding partners and thereby affect cellular functions. Taken together, our results point to a previously unrecognized change in the Lamin A/C Ig-fold that affects recognition by the JOL-2 antibody. These findings are not only important for the interpretation of immunofluorescence data for Lamin A/C, but also raise the intriguing prospect that the conformational changes may play a role in Lamin A/C mediated cellular function.

cell biology↗

bioRxiv2022

A widespread methylotroph acyl-homoserine lactone synthase produces an atypical quorum sensing signal

Pink pigmented facultative methylotrophs of the genera Methylorubrum and Methylobacterium are omnipresent bacteria often found associated with plants. Despite their widespread occurrence, the molecular details of how these organisms interact with each other and their environment remain understudied. We analyzed genes encoding N-acylhomoserine lactone quorum sensing signal synthases in published genomes of these bacteria and determined that the product of the largest group of signal synthases had not been characterized. We subsequently identified this N-acylhomoserine lactone product using inverse stable isotopic labeling (InverSIL), which revealed an atypical signal. We then demonstrate in one representative strain that this signal activates its cognate LuxR-family transcription factor and is produced in a positive feedback loop. These results reveal a previously undescribed yet widespread signal used by pink pigmented facultative methylotrophs, which helps us understand the chemical ecology of these important bacteria.

microbiology↗

bioRxiv2022

Increasing Muscle Hypertrophy with a Natural Product Designed to Inhibit SIRT1

Muscle mass and strength are predictors of longevity. We have previously identified a series of molecular brakes that slow muscle growth in response to stress. One potential stress that we hypothesized would limit muscle growth is caloric stress through the activation of SIRT1. We therefore identified natural product inhibitors of SIRT1 and tested their effects on load-induced increases in muscle fiber cross-sectional area (fCSA) using an incomplete factorial design. Supplying varying amounts of three natural products for the full two-week period of overload resulted in increases in fCSA that varied from -2 to 113%. Using these data, we produced a model that predicted the optimal combination and concentration of each natural product and validated this model in a separate cohort of animals. Following two weeks of overload, fCSA in the optimal group increased 62%, whereas in the placebo fCSA increased only 3%. The greater increase in fCSA was not the result of an increase in ribosomal mass. In fact, the optimal group showed significantly less of the 5 external transcribed spacer, a marker of 47S ribosomal RNA synthesis, and a trend for decreased total RNA. In spite of the lower ribosome mass, the increase in protein synthesis was similar, suggesting that the natural product cocktail may be increasing ribosomal efficiency rather than capacity. These data suggest that inhibition of SIRT1, together with exercise, may be useful in increasing muscle fCSA.

physiology↗

bioRxiv2022

Adipocytes reprogram carbon and nitrogen metabolism to maintain lipogenic flux in the absence of Bckdha

Dysregulated branched chain amino acid (BCAA) metabolism has emerged as a key metabolic feature associated with the obese insulin resistant state, and adipose BCAA catabolism is decreased in this context. BCAA catabolism is upregulated early in adipogenesis, but the impact of suppressing this pathway on the broader metabolic functions of the resultant adipocyte remain unclear. Here, we use CRISPR/Cas9 to target Bckdha and Acad8 in pre-adipocytes and induce a deficiency in BCAA or valine catabolism through differentiation. We characterise the transcriptional and metabolic phenotype of these cells using RNAseq and 13C metabolic flux analysis within a network spanning glycolysis, tricarboxylic (TCA) acid metabolism, BCAA catabolism, and fatty acid synthesis. While lipid droplet accumulation is maintained in Bckdha-deficient adipocytes, they display a more fibroblast-like transcriptional signature. In contrast, Acad8 deficiency minimally impacts gene expression. Decreased glycolytic flux emerges as the most distinct metabolic feature of Bckdha-deficient cells, accompanied by a [~]40% decrease in lactate secretion, yet pyruvate oxidation and utilization for de novo lipogenesis are increased to compensate for loss of BCAA carbon. Glutamine anaplerosis was also increased, though we observed a general decrease in levels of most non-essential amino acids consistent with an impact on nitrogen homeostasis. Overall, our data suggest that both metabolic and regulatory cross-talk exists between BCAA catabolism, glycolysis, and nitrogen metabolism in differentiated adipocytes. Suppression of BCAA catabolism associated with metabolic syndrome may result in a metabolically compromised adipocyte.

cell biology↗