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BioRxiv : the Preprint Server For... Jun 2024Membrane potential (MP) changes can provide a simple readout of bacterial functional and metabolic state or stress levels. While several optical methods exist for...
Membrane potential (MP) changes can provide a simple readout of bacterial functional and metabolic state or stress levels. While several optical methods exist for measuring fast changes in MP in excitable cells, there is a dearth of such methods for absolute and precise measurements of steady-state membrane potentials (MPs) in bacterial cells. Conventional electrode-based methods for the measurement of MP are not suitable for calibrating optical methods in small bacterial cells. While optical measurement based on Nernstian indicators have been successfully used, they do not provide absolute or precise quantification of MP or its changes. We present a novel, calibrated MP recording approach to address this gap. Our method is based on (i) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer (PeT) and (ii) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily recorded, quantified and visualized. Using our preliminary -specific MP versus VF lifetime calibration, we estimated the MP for unperturbed cells to be -65 mV and that for chemically depolarized cells as -14 mV. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research.
PubMed: 38915670
DOI: 10.1101/2024.06.13.598880 -
BioRxiv : the Preprint Server For... Jun 2024Socially coordinated threat responses support the survival of animal groups. Given their distinct social roles, males and females must differ in such coordination. Here,...
Socially coordinated threat responses support the survival of animal groups. Given their distinct social roles, males and females must differ in such coordination. Here, we report such differences during the synchronization of auditory-conditioned freezing in mouse dyads. To study the interaction of emotional states with social cues underlying synchronization, we modulated emotional states with prior stress or modified the social cues by pairing unfamiliar or opposite-sex mice. In same-sex dyads, males exhibited more robust synchrony than females. Stress disrupted male synchrony in a prefrontal cortex-dependent manner but enhanced it in females. Unfamiliarity moderately reduced synchrony in males but not in females. In dyads with opposite-sex partners, fear synchrony was resilient to both stress and unfamiliarity. Decomposing the synchronization process in the same-sex dyads revealed sex-specific behavioral strategies correlated with synchrony magnitude: following partners' state transitions in males and retroacting synchrony-breaking actions in females. Those were altered by stress and unfamiliarity. The opposite-sex dyads exhibited no synchrony-correlated strategy. These findings reveal sex-specific adaptations of socio-emotional integration defining coordinated behavior and suggest that sex-recognition circuits confer resilience to stress and unfamiliarity in opposite-sex dyads.
PubMed: 38915653
DOI: 10.1101/2024.06.09.598132 -
BioRxiv : the Preprint Server For... Jun 2024ATP-citrate lyase (ACLY) converts citrate into acetyl-CoA and oxaloacetate in the cytosol. It plays a prominent role in lipogenesis and fat accumulation coupled to...
BACKGROUND
ATP-citrate lyase (ACLY) converts citrate into acetyl-CoA and oxaloacetate in the cytosol. It plays a prominent role in lipogenesis and fat accumulation coupled to excess glucose, and its inhibition is approved for treating hyperlipidemia. In RNAseq analysis of human failing myocardium, we found ACLY gene expression is reduced; however the impact this might have on cardiac function and/or metabolism has not been previously studied. As new ACLY inhibitors are in development for cancer and other disorders, such understanding has added importance.
METHODS
Cardiomyocytes, ex-vivo beating hearts, and in vivo hearts with ACLY inhibited by selective pharmacologic (BMS303141, ACLYi) or genetic suppression, were studied. Regulation of ACLY gene/protein expression, and effects of ACLYi on function, cytotoxicity, tricarboxylic acid (TCA)-cycle metabolism, and redox and NAD+/NADH balance were assessed. Mice with cardiac ACLY knockdown induced by AAV9-acly-shRNA or cardiomyocyte tamoxifen-inducible Acly knockdown were studied.
RESULTS
Acly gene expression was reduced more in obese patients with heart failure and preserved EF (HFpEF) than HF with reduced EF. In vivo pressure-overload and in vitro hormonal stress increased ACLY protein expression, whereas it declined upon fatty-acid exposure. Acute ACLYi (1-hr) dose-dependently induced cytotoxicity in adult and neonatal cardiomyocytes, and caused substantial reduction of systolic and diastolic function in myocytes and ex-vivo beating hearts. In the latter, ATP/ADP ratio also fell and lactate increased. U13C-glucose tracing revealed an ACLYdependent TCA-bypass circuit in myocytes, where citrate generated in mitochondria is transported to the cytosol, metabolized by ACLY and then converted to malate to re-enter mitochondria,bypassing several NADH-generating steps. ACLYi lowered NAD+/NADH ratio and restoring this balance ameliorated cardiomyocyte toxicity. Oxidative stress was undetected with ACLYi. Adult hearts following 8-weeks of reduced cardiac and/or cardiomyocyte ACLY downregulation exhibited ventricular dilation and reduced function that was prevented by NAD augmentation. Cardiac dysfunction from ACLY knockdown was worse in hearts subjected to sustained pressureoverload, supporting a role in stress responses.
CONCLUSIONS
ACLY supports normal cardiac function through maintenance of the NAD+/NADH balance and is upregulated by hemodynamic and hormonal stress, but depressed by lipid excess. ACLY levels are most reduced in human HFpEF with obesity potentially worsening cardio-metabolic reserve.
PubMed: 38915649
DOI: 10.1101/2024.06.09.598152 -
BioRxiv : the Preprint Server For... Jun 2024The kidney filters nutrient waste and bodily fluids from the bloodstream, in addition to secondary functions of metabolism and hormone secretion, requiring an...
UNLABELLED
The kidney filters nutrient waste and bodily fluids from the bloodstream, in addition to secondary functions of metabolism and hormone secretion, requiring an astonishing amount of energy to maintain its functions. In kidney cells, mitochondria produce adenosine triphosphate (ATP) and help maintain kidney function. Due to aging, the efficiency of kidney functions begins to decrease. Dysfunction in mitochondria and cristae, the inner folds of mitochondria, is a hallmark of aging. Therefore, age-related kidney function decline could be due to changes in mitochondrial ultrastructure, increased reactive oxygen species (ROS), and subsequent alterations in metabolism and lipid composition. We sought to understand if there is altered mitochondrial ultrastructure, as marked by 3D morphological changes, across time in tubular kidney cells. Serial block facing-scanning electron microscope (SBF-SEM) and manual segmentation using the Amira software were used to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented, compared to the 3-month, with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we show that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney due to aging. Disruption of the MICOS complex shows altered mitochondrial calcium uptake and calcium retention capacity, as well as generation of oxidative stress. We found significant, detrimental structural changes to aged kidney tubule mitochondria suggesting a potential mechanism underlying why kidney diseases occur more readily with age. We hypothesize that disruption in the MICOS complex further exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, thus impacting kidney health.
TRANSLATIONAL STATEMENT
Due to aging, the efficiency of kidney functions begins to decrease and the risk of kidney diseases may increase, but specific regulators of mitochondrial age-related changes are poorly explained. This study demonstrates the MICOS complex may be a target for mitigating age-related changes in mitochondria. The MICOS complex can be associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies.
PubMed: 38915644
DOI: 10.1101/2024.06.09.598108 -
BioRxiv : the Preprint Server For... Jun 2024The fungus is an opportunistic pathogen of people that reprograms its translatome to facilitate adaptation and virulence within the host. We studied the role of...
UNLABELLED
The fungus is an opportunistic pathogen of people that reprograms its translatome to facilitate adaptation and virulence within the host. We studied the role of Hog1/p38 in reprogramming translation during thermal stress adaptation, and found that this pathway acts on translation via crosstalk with the Gcn2 pathway, a well-studied regulator of general translation control. Using a combination of molecular assays and phenotypic analysis, we show that increased output from the Gcn2 pathway in a Hog1 deletion mutant is associated with rescue of thermal stress adaptation at both molecular and phenotypic scales. We characterize known outputs of the Hog1 pathway during thermal stress as either Gcn2-dependent or Gcn2-independent, and demonstrate that Hog1 activation regulates the Gcn2 pathway even in the absence of thermal stress. Finally, we implicate this phenomenon in another Hog1-regulated process, morphogenesis, and recapitulate Hog1-Gcn2 crosstalk in the distantly related fungal pathogen, Our results point to an important link between the stress response machinery and translation control, and clarify the etiology of phenotypes associated with Hog1 deletion. More broadly, this study highlights complex interplay between core conserved signal transduction pathways and the utility of molecular assays to better understand how these pathways are connected.
IMPORTANCE
is an opportunistic pathogen of people that causes deadly cryptococcal meningitis, which is is responsible for an estimated 19% of AIDS-related mortality. When left untreated, cryptococcal meningitis is uniformly fatal, and in patients receiving the most effective antifungal regimens, mortality remains high. Thus, there is a critical need to identify additional targets that play a role in adaptation to the human host and virulence. This study explores the role of the stress response kinases Hog1 and Gcn2 in thermoadaptation, which is pre-requisite for virulence. Our results show that compensatory signaling occurs via the Gcn2 pathway when Hog1 is deleted, and that disruption of both pathways increases sensitivity to thermal stress. Importantly, our study highlights the insufficiency of using single gene deletion mutants to study gene function, since many phenotypes associated with Hog1 deletion were driven by Gcn2 signaling in this background, rather than loss of direct Hog1 activity.
PubMed: 38915642
DOI: 10.1101/2024.06.11.598457 -
BioRxiv : the Preprint Server For... Jun 2024Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases,...
Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic, stress-independent activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic, stress-independent activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that stress-independent activation of these ISR kinases reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic, stress-independent activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.
PubMed: 38915623
DOI: 10.1101/2024.06.10.598126 -
BioRxiv : the Preprint Server For... Jun 2024Diffuse midline gliomas (DMGs) are lethal primary brain tumors in children. The imipridones ONC201 and ONC206 induce mitochondrial dysfunction and have emerged as...
UNLABELLED
Diffuse midline gliomas (DMGs) are lethal primary brain tumors in children. The imipridones ONC201 and ONC206 induce mitochondrial dysfunction and have emerged as promising therapies for DMG patients. However, efficacy as monotherapy is limited, identifying a need for strategies that enhance response. Another hurdle is the lack of biomarkers that report on drug-target engagement at an early timepoint after treatment onset. Here, using H-magnetic resonance spectroscopy, which is a non-invasive method of quantifying metabolite pool sizes, we show that accumulation of ψ-aminobutyric acid (GABA) is an early metabolic biomarker that can be detected within a week of ONC206 treatment, when anatomical alterations are absent, in mice bearing orthotopic xenografts. Mechanistically, imipridones activate the mitochondrial protease ClpP and upregulate the stress-responsive transcription factor ATF4. ATF4, in turn, upregulates glutamate decarboxylase, which synthesizes GABA, and downregulates , which degrades GABA, leading to GABA accumulation in DMG cells and tumors. Functionally, GABA secreted by imipridone-treated cells acts in an autocrine manner via the GABAB receptor to induce expression of superoxide dismutase (SOD1), which mitigates imipridone-induced oxidative stress and, thereby, curbs apoptosis. Importantly, blocking autocrine GABA signaling using the clinical stage GABAB receptor antagonist SGS-742 exacerbates oxidative stress and synergistically induces apoptosis in combination with imipridones in DMG cells and orthotopic tumor xenografts. Collectively, we identify GABA as a unique metabolic adaptation to imipridones that can be leveraged for non-invasive assessment of drug-target engagement and therapy. Clinical translation of our studies has the potential to enable precision metabolic therapy and imaging for DMG patients.
ONE SENTENCE SUMMARY
Imipridones induce GABA accumulation in diffuse midline gliomas, an effect that can be leveraged for therapy and non-invasive imaging.
PubMed: 38915617
DOI: 10.1101/2024.06.07.597982 -
BioRxiv : the Preprint Server For... Jun 2024Telomeric DNA, composed of short, direct repeats, is of crucial importance for chromosome stability. Due to intrinsic problems with replicating this DNA, the repeat...
Telomeric DNA, composed of short, direct repeats, is of crucial importance for chromosome stability. Due to intrinsic problems with replicating this DNA, the repeat tracts shorten at each cell division. Once repeat tracts become critically short, a telomeric stress signal induces cellular senescence and division arrest, which eventually may lead to devastating age-related degenerative diseases associated with dysfunctional telomers. Conversely, maintenance of telomere length by telomerase upregulation is a hallmark of cancer. Therefore, telomere length is a critical determinant of telomere function. How telomere length is established and molecular mechanisms for telomere-specific length regulation remained unknown. Here we show that subtelomeric chromatin is a determinant for how telomere equilibrium set-length is established in . The results demonstrate that telomerase recruitment mediated by the telomere-associated Sir4 protein is modulated on chromosome 3L in a telomere-specific way. Increased Sir4 abundance on subtelomeric heterochromatin of this specific telomere leads to telomere lengthening of only that telomere in , but not at other telomeres. Therefore, this work describes a mechanism for a how telomere-specific repeat tract length can be established. Further, our results will force the evaluation of telomere length away from a generalized view to a more telomere-specific consideration.
PubMed: 38915611
DOI: 10.1101/2024.06.12.598646 -
BioRxiv : the Preprint Server For... Jun 2024Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse...
Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse models. Previous work supports that promotes longevity, stress resistance, and healthspan by rewiring endogenous metabolism. However, there are five FMOs and five mammalian FMOs, and it is not known whether promoting longevity and health benefits is a conserved role of this gene family. Here, we report that expression of promotes lifespan extension and paraquat stress resistance downstream of both dietary restriction and inhibition of mTOR. We find that overexpression of in just the hypodermis is sufficient for these benefits, and that this expression significantly modifies the transcriptome. By analyzing changes in gene expression, we find that genes related to calcium signaling are significantly altered downstream of expression. Highlighting the importance of calcium homeostasis in this pathway, overexpressing animals are sensitive to thapsigargin, an ER stressor that inhibits calcium flux from the cytosol to the ER lumen. This calcium/ interaction is solidified by data showing that modulating intracellular calcium with either small molecules or genetics can change expression of and/or interact with to affect lifespan and stress resistance. Further analysis supports a pathway where modulates calcium homeostasis downstream of activating transcription factor-6 ( ), whose knockdown induces and requires expression. Together, our data identify as a longevity- promoting gene whose actions interact with known longevity pathways and calcium homeostasis.
PubMed: 38915593
DOI: 10.1101/2024.05.17.594584 -
BioRxiv : the Preprint Server For... Jun 2024Black yeasts and relatives comprise Micro-Colonial Fungi (MCFs) which are slow-growing stress-tolerant micro-eukaryotes that specialize in extreme environments. MCFs are...
Black yeasts and relatives comprise Micro-Colonial Fungi (MCFs) which are slow-growing stress-tolerant micro-eukaryotes that specialize in extreme environments. MCFs are paraphyletic and found in the Orders ( ) and ( ). We have isolated and described three new MCFs species from desert biological soil crusts (BSCs) collected from two arid land regions: Joshua Tree National Park (Mojave Desert) and UC Natural Reserve at Boyd Deep Canyon (confluence of Mojave and Sonoran Deserts). BSCs are composite assemblages of cyanobacteria, eukaryotic algae, fungi, lichens, and bryophytes embedded into the surface of desert soils, providing a protective buffer against the harsh desert environment. Our work focused on one type of desert BSC, the cyanolichen crust dominated by Using culture-dependent protocols, three MCFs were axenically isolated from their respective samples along with the extracted DNA. Their genomes were sequenced using Illumina and Nanopore, and finally assembled and annotated using hybrid assembly approaches and established bioinformatics pipelines to conduct final taxonomic phylogenetic analysis and placement. The three species described here are unique specimen from desert BSCs, here we introduce, ( , ( ), and ( ).
PubMed: 38915581
DOI: 10.1101/2024.06.12.598762