Biology subjects

Garcia, A.

Publications and source records attributed to Garcia, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Clonal analysis of SepSecS-specific CD4 T cells reveals a new HLA-DPA102:01/HLA-DPB101:01-restricted immunodominant epitope in autoimmune hepatitis

Autoreactive CD4 T cells, recognizing liver-self-antigens such as SepSecS, are main drivers of the chronic inflammatory response during autoimmune hepatitis (AIH). Previous studies have uncovered immunodominant SepSecS epitopes often associated with HLA-DRB1*03 or HLA-DRB1*04 restriction, two alleles enriched in AIH population. However, HLA restriction of numerous SepSecS epitopes remains incomplete and it is still unclear if immunodominant epitopes could be presented by non-HLA-DR molecules. Here, we investigated epitope recognition of SepSecS-specific TCRs isolated from AIH patients, and their HLA restriction by generating TCR hybridoma cell lines. Seventeen TCRs recognized eight SepSecS epitopes with four distinct HLA restrictions, including a novel HLA-DPA1*02:01/DPB1*01:01-restricted SepSecS epitope. TCR clustering analysis using GLIPH2 algorithm suggested that this epitope is recognized by multiple distinct TCRs in HLA-DPA1*02:01[~]HLA-DPB1*01:01 patients. Our study provides new insights into liver-self-antigen T cell reactivity during AIH, which could offer potential therapeutic strategies by targeting autoreactive CD4 T cells.

immunology↗

bioRxiv2025

Dual Mechanisms for Heterogeneous Responses of Inspiratory Neurons to Noradrenergic Modulation

Respiration is an essential involuntary function necessary for survival. This poses a challenge for the control of breathing. The preBotzinger complex (preBotC) is a heterogeneous neuronal network responsible for driving the inspiratory rhythm. While neuromodulators such as norepinephrine (NE) allow it to be both robust and flexible for all living beings to interact with their environment, the basis for how neuromodulation impacts neuron-specific properties remains poorly understood. In this work, we examine how NE influences different preBotC neuronal subtypes by modeling its effects through modulating two key parameters: calcium-activated nonspecific cationic current gating conductance (gCAN) and inositol-triphosphate (IP3), guided by experimental studies. Our computational model captures the experimentally observed differential effects of NE on distinct preBotC bursting patterns. We show that this dual mechanism is critical for inducing conditional bursting and identify specific parameter regimes where silent neurons remain inactive in the presence of NE. Furthermore, using methods of dynamical systems theory, we uncover the mechanisms by which NE differentially modulates burst frequency and duration in NaP-dependent and CAN-dependent bursting neurons. These results align well with previously reported experimental findings and provide a deeper understanding of cell-specific neuromodulatory responses within the respiratory network. MSC codes37N25, 34C23, 34C60, 34E13, 34E15, 92C20

neuroscience↗

bioRxiv2025

Comparative genomics-driven design of virus-delivered short RNA inserts triggering robust gene silencing

Engineered RNA viruses can downregulate target genes through virus-induced gene silencing (VIGS). Largely optimized using the model plant Nicotiana benthamiana, current VIGS protocols rely on viral vectors engineered to deliver 200-400 nt inserts with homology to a target gene, often located in less conserved regions to ensure specificity. Given the N. benthamiana allopolyploidy and functional redundancy, we leveraged enhanced genomics and transcriptomics resources to obtain curated locus annotations, which guided the design of virus-delivered short RNA inserts (vsRNAi) for simultaneous targeting of homeologous gene pairs. Our results showed that quantitative silencing phenotypes could be obtained using 24-, 28-, and 32-nt vsRNAi that targeted conserved regions of functionally redundant gene pairs. vsRNAi triggered informative transcriptome-wide changes, and target transcript downregulation was linked to gene-pair-specific production of 21- and 22-nt sRNAs. The portability of vsRNAi to crops was confirmed in tomato (Solanum lycopersicum) and scarlet eggplant (Solanum aethiopicum). Overall, our results support the usefulness of vsRNAi-based approaches in functional genomics and trait reprogramming of the model species N. benthamiana, as well as of crops that contribute to global and local food provisions. HIGHLIGHTS O_LIComparative genomics enables design of virus-delivered short RNA inserts (vsRNAi) C_LIO_LI32-, 28-, and 24-nt vsRNAi resulted in quantitative gene silencing phenotypes C_LIO_LIvsRNAi trigger transcriptome-wide changes for gene functional characterization C_LIO_LIvsRNAi elicit production of 21- and 22-nt small RNAs and gene downregulation C_LIO_LIvsRNAi approaches assist functional genomics and phenotypic alteration in crops C_LI

plant biology↗

bioRxiv2025

What determines trophic niche breadth? A global analysis of freshwater fishes using isospaces

Trophic niche is one of the most tractable dimensions of a species niche. Several hypotheses have been proposed to explain global variation in species trophic niches, but empirical tests are limited. Stable isotope analysis (SIA) of {delta}{superscript 1}3C (a proxy for basal sources) and {delta}{superscript 1} N (a proxy for vertical trophic position) has increasingly been used to estimate trophic niche variation. We performed SIA on a dataset of over 33,000 stable isotope samples from freshwater fishes across six ecoregions. For 541 populations (358 species), we estimated the size of {delta}{superscript 1}3C x {delta}{superscript 1} N isospace--a bivariate representation of trophic niche breadth. We evaluated isospace variation in relation to environmental factors, species traits, and sampling scale, testing both novel and long-standing hypotheses about the drivers of trophic niche variation. A Boosted Regression Tree Model best predicted isospace, with environmental and trait-based variables among the strongest predictors. Fish isospaces were broader in regions with warmer temperatures, higher humidity, and precipitation variability, suggesting that more productive, diverse, and seasonal ecosystems are associated with broader trophic niches. Conversely, isospace size declined with increasing basin fish richness, which may reflect heightened interspecific competition or historical competitive exclusion. Isospace size was slightly smaller in large lakes compared to rivers, streams, and small lakes. Within-population variation in body size was strongly and positively associated with isospace size, highlighting the role of ontogenetic dietary shifts. Fish with truncate-rounded fins had broader isospaces than those with forked-lunate fins, likely due to the former having more limited movement and greater spatial isolation that resulted in greater between-individual niche variation. Primary consumers had larger isospaces than intermediate and top consumers. Predators showed relationships consistent those observed from analysis encompassing all trophic guilds, but non-predators deviated with regards to four variables--solar radiation, basin richness, average body size, and caudal fin aspect ratio. Isospace size increased with the number of sampled individuals, habitats and years surveyed. Although isospace patterns have well-documented limitations as trophic ecology metrics, our findings nonetheless conform with several longstanding hypotheses for trophic niche variation and stimulate new ideas about environmental and biological drivers of niche breadth.

ecology↗

bioRxiv2024

Experimental Phage Evolution Results in Expanded Host Ranges Against MDR and XDR Klebsiella pneumoniae Isolates

Resistance to antibiotics is approaching crisis levels for organisms such as the ESKAPEE pathogens (includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) that often are acquired in hospitals. These organisms sometimes have acquired plasmids that confer resistance to most if not all beta-lactam antibiotics such as those produced by Carbapenem Resistant Enterobacterales (CREs). We have been developing alternative means for dealing with antibiotic resistant microbes that cause infections in humans by developing viruses (bacteriophages) that attack and kill them. We have been working with one of the ESKAPEE pathogens, K. pneumoniae, that has one of the highest propensities for antimicrobial resistance, to develop phages that target and kill it. We identified a number of phages that have lytic capacity against only a few clinical isolates, and through experimental evolution over the course of 30 days, were able to vastly expand the host ranges of these phages to kill a broader range of clinical K. pneumoniae isolates including MDR (multi-drug resistant) and XDR (extensively-drug resistant) isolates. Most interestingly, they were capable of inhibiting growth of clinical isolates both on solid and in liquid medium over extended periods. That we were able to extend the host ranges of multiple naive MDR and XDR K. pneumoniae through experimental phage evolution suggests that such a technique may be applicable to other antibiotic-resistant organisms to help stem the tide of antibiotic resistance and offer further options for medical treatments. ImportanceBacterial pathogens are becoming greater threats given the rise in antibiotic resistance, where traditional therapies may no longer work to cure some infections. Chief amongst these multidrug resistant infections (MDR) and extensively drug-resistant infections (XDR) is Klebsiella pneumoniae, which is known to sometimes harbor genetic elements that render it incredibly difficult to treat with conventional antibiotics. Treatments like bacteriophages have not had much success against such pathogens because resistance to the phages used often develops rapidly. We adapted a co-evolutionary technique to develop K. pneumoniae phages to be highly active longitudinally against K. pneumoniae clinical isolates. In as few as 30 days, we were able to vastly expand the host ranges of K. pneumoniae phages against MDR and XDR clinical isolates and that maintain their infectivity over clinically relevant time periods. By adapting these established techniques to clinical MDR and XDR K. pneumoniae isolates, we believe we can establish similar techniques for expanding phage host ranges against most antibiotic-resistant bacteria. As such, phages can be viable alternatives to antibiotics when antibiotic resistance exists in hospitals and communities.

microbiology↗

bioRxiv2024

Cooperative Role of PACT and ADAR1 in Preventing Aberrant PKR Activation by Self-Derived Double-Stranded RNA

Double-stranded RNAs (dsRNAs) produced during viral infections are recognized by the innate immune sensor protein kinase R (PKR), triggering a host translation shutoff that inhibits viral replication and propagation. Given the harmful effects of uncontrolled PKR activation, cells must tightly regulate PKR to ensure that its activation occurs only in response to viral infections, not endogenous dsRNAs. Here, we use CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that exploits translation levels as a readout and identifies PACT as a key inhibitor of PKR during viral infection. We find that cells deficient for PACT hyperactivate PKR in response to several different RNA viruses, raising the question of why cells need to limit PKR activity. Our results demonstrate that PACT cooperates with ADAR1 to suppress PKR activation from self-dsRNAs in uninfected cells. The simultaneous deletion of PACT and ADAR1 results in synthetic lethality, which can be fully rescued in PKR-deficient cells. We propose that both PACT and ADAR1 act as essential barriers against PKR, creating a threshold of tolerable levels to endogenous dsRNA in cells without activating PKR-mediated translation shutdown and cell death.

microbiology↗

bioRxiv2024

A lipid transport Mla Pqi Chimeric system is essential for Brucella abortus survival in macrophages

The envelope of diderm bacteria comprises of an inner membrane (IM) and an outer membrane (OM). Several pathways have been recently identified that facilitate the transport of phospholipids between the two membranes in Escherichia coli, including maintaining OM lipid asymmetry (Mla) and paraquat inducible (Pqi) systems. In this study, we report the identification and the characterization of a complex named Mpc in the intracellular pathogen Brucella abortus. Mpc is conserved in numerous species of Hyphomicrobiales and exhibits homology to both the Mla and Pqi systems. Mpc is essential for bacterial growth under conditions of envelope stress and for survival within macrophages during the early stages of infection. Analyses of protein-protein interactions and structural predictions indicate that the Mpc complex bridges IM to OM. The absence of this system results in an altered lipid composition of the OM vesicles, supporting the fact that Mpc plays a role in the transport of lipids between membranes. The discovery of a novel lipid trafficking system enhances the diversity and complexity of known lipid trafficking systems within diderm bacteria.

microbiology↗

bioRxiv2024

A sensory approach to turbidity: How sources and levels shape aquatic light environments and fish visual ecology

Turbidity is a ubiquitous source of sensory pollution that is likely to impact the appearance of the visual stimuli that animals rely on for survival and reproduction. Understanding how different turbidity sources impact the appearance of the ambient light environment is the foundational first step towards predicting whether and how animals will cope with the global increases in the severity and frequency of high turbidity events caused by anthropogenic disturbance. Here, we measured how four common turbidity sources (algae, bentonite, calcium carbonate, and kaolin), and variable turbidity levels, changed the appearance of the ambient light environment. We measured total number of photons (luminance), hue, chroma, and image contrast, and we evaluated the effect of each turbidity source and level on settling rate, pH, and KH. Both turbidity source and turbidity level impacted the appearance of the ambient light environment. With increasing turbidity level, calcium carbonate and kaolin increased luminance while algae decreased luminance, bentonite caused the greatest change in hue, and algae caused the greatest change in chroma. This demonstrates that the impacts of different turbidity sources on the ambient light environment are not uniform, giving a potential explanation for the discrepancies between studies on the effects of turbidity on fish behaviour. Consideration of the effect of specific turbidity sources on ambient light is crucial for the design of experiments that seek to investigate how changes in turbidity impact the perception of important visual information, which underpins the survival and reproductive success of aquatic organisms around the world. Summary StatementDifferent turbidity sources and levels uniquely affect aquatic light environments and fish visual perception, emphasizing the need for these factors when interpreting fish behaviour and the ecological consequences of turbidity.

animal behavior and cognition↗

bioRxiv2024

Synthesis of Novel N-Substituted β-Amino Acid Derivatives Bearing 2-Hydroxyphenyl Moieties as Promising Antimicrobial Candidates Targeting Multidrug-Resistant Gram-Positive Pathogens

The increasing prevalence of antimicrobial resistance among ESKAPE group pathogens presents a significant challenge in the healthcare sector, contributing to higher morbidity and mortality rates globally. Thus, it is essential to develop novel antimicrobial agents effective against drug-resistant pathogens. In this study, we report the synthesis and in vitro antimicrobial activity characterization of novel N-substituted {beta}-amino acid derivatives bearing 2-hydroxyphenyl core against multidrug-resistant bacterial pathogens. The synthesized compounds (2-26) exhibited promising antimicrobial activity specifically against Gram-positive bacteria, with minimum inhibitory concentrations (MIC) ranging from 4 to 128 {micro}g/mL. None of the compounds demonstrated activity against Gram-negative pathogens or drug-resistant fungi. Compounds 9 (R = 4-nitrophenyl), 17 (R = 5-nitro-2-thienyl), 18 (R = 5-nitro-2-furyl), thiosemicarbazide 16, and 26 exhibited the most promising activity against Staphylococcus aureus MRSA USA300 lineage strain TCH-1516, with MIC values between 4 and 16 {micro}g/mL. Compound 26 demonstrated strong antimicrobial activity against both S. aureus TCH-1516 and E. faecalis AR-0781, with the activity comparable to control antibiotics. These findings indicate that N-substituted {beta}-amino acid derivatives with a 2-hydroxyphenyl core warrant further investigation as a potential scaffold for the further development of antimicrobial agents based on compound 26 targeting Gram-positive pathogens.

microbiology↗

bioRxiv2024

Neurovascular Impulse Response Function (IRF) during spontaneous activity differentially reflects intrinsic neuromodulation across cortical regions

Ascending neuromodulatory projections from deep brain nuclei generate internal brain states that differentially engage specific neuronal cell types. Because neurovascular coupling is cell-type specific and neuromodulatory transmitters have vasoactive properties, we hypothesized that the impulse response function (IRF) linking spontaneous neuronal activity with hemodynamics would depend on neuromodulation. To test this hypothesis, we used optical imaging to measure (1) release of neuromodulatory transmitters norepinephrine (NE) or acetylcholine (ACh), (2) Ca2+ activity of local cortical neurons, and (3) changes in hemoglobin concentration and oxygenation across the dorsal surface of cerebral cortex during spontaneous neuronal activity in awake mice. A canonical convolution model with a global, stationary IRF (i.e., the convolution kernel) describing evolution of total hemoglobin (HbT, reflective of dilation dynamics) with respect to Ca2+, resulted in a poor fit to the data. However, the HbT time-course was well predicted, pixel-by-pixel, by a weighted sum of Ca2+ and NE time-courses. Consistent with this result, modeling HbT as a weighted sum of stationary Ca2+ - and NE-specific IRFs (IRFCa2+ and IRFNE) convolved with the respective time-courses dramatically improved the fit compared to the global IRF. IRFCa2+ and IRFNE, estimated from the data, were positive and negative, respectively. In contrast to NE, ACh was largely redundant with Ca2+ and therefore did not improve HbT estimation. Because NE covaried with arousal, we observed instances of the diminished hemodynamic coherence between cortical regions during high arousal despite coherent behavior of the underlying neuronal Ca2+ activity. We conclude that while neurovascular coupling with respect to neuronal Ca2+ is a dynamic and seemingly complex phenomenon, hemodynamic fluctuations can be captured by a simple linear model with stationary IRFs with respect to the underlying dilatory and constrictive forces. In the current study, these forces were captured by the positive IRFCa2+ (dilation) and negative IRFNE (constriction). Without accounting for NE neuromodulation and the associated vasoconstriction, diminished hemodynamic coherence, commonly referred to as "functional (dys)connectivity" in BOLD fMRI studies, can be falsely interpreted as neuronal desynchronizations.

neuroscience↗

bioRxiv2024

Nano-org, a functional resource for single-molecule localisation microscopy data

We present a publicly accessible, curated, and functional resource, termed "nano-org", containing single-molecule localisation microscopy (SMLM) data representing the nanoscale distributions of proteins in cells. Nano-org is searchable by comparing the statistical similarity of the datasets it contains. This unique functionality allows the resource to be used to understand the relationships of nanoscale architectures between proteins, cell types or conditions, enabling a new field of spatial nano-omics.

biophysics↗

bioRxiv2024

TAM receptors mediate the Fpr2-driven pain resolution and fibrinolysis after nerve injury

Nerve injury causes neuropathic pain and multilevel nerve barrier disruption. Nerve barriers consist of perineurial, endothelial, and myelin barriers. So far, it is unclear whether resealing nerve barriers fosters pain resolution and recovery. To this end, we analysed the nerve barrier property portfolio, pain behaviour battery, and lipidomics for precursors of specialized pro-resolving meditators (SPMs) and their receptors in chronic constriction injury of rat sciatic nerve to identify targets for pain resolution by resealing the selected nerve barriers. Of the three nerve barriers - perineurium, capillaries, and myelin - only capillary tightness specifically against larger molecules, such as fibrinogen, recuperated with pain resolution. Fibrinogen immunoreactivity was not only elevated in rats at the time of neuropathic pain but also in nerve biopsies from patients with (but not without) painful polyneuropathy indicating that sealing of the vascular barrier might be novel approach in pain treatment. 15R-HETE (hydroxyeicosatetraenoic acid), a precursor of aspirin-triggered lipoxin A4, were specifically upregulated at the beginning of pain resolution. Repeated local application of resolvin D1-laden nanoparticles or Fpr2 agonists sex-independently resulted in accelerated pain resolution and fibrinogen removal. Clearing macrophages (Cd206) and fibrinolytic pathways (Plat) were also induced while inflammation (Tnf) and inflammasomes (Nlrp3) were unaffected by this treatment. Blocking TAM receptors (Tyro3, Axl, and Mer) and tyrosine kinase receptors linking haemostasis and inflammation completely inhibited all the effects. In summary, nanoparticles can be used as transporters for fleeting lipids, such as SPMs, and therefore expand the array of possible therapeutic agents. Thus, the Fpr2-Cd206-TAM receptor axis may be a suitable target for strengthening the capillary barrier, removing endoneurial fibrinogen, and boosting pain resolution in patients with chronic neuropathic pain.

neuroscience↗

bioRxiv2024

Contribution of telacebec to novel drug regimens in a murine tuberculosis model

The clinical efficacy of combination drug regimens containing the first generation diarylquinoline (DARQ) bedaquiline in the treatment of multidrug-resistant tuberculosis has validated ATP synthesis as a vulnerable pathway in Mycobacterium tuberculosis. New DARQs in clinical development may be even more effective than bedaquiline, including against emerging bedaquiline-resistant strains. Telacebec (T) is a novel cytochrome bc1:aa3 oxidase inhibitor that also inhibits ATP synthesis. Based on its demonstrated efficacy as a monotherapy in mice and in a phase 2a clinical trial, we used an established BALB/c mouse model of tuberculosis (TB) to test the contribution of T to novel combination therapies against two strains of M. tuberculosis (H37Rv and HN878) in an effort to find more effective regimens. Overall, T was more effective in regimens against the HN878 strain than against the H37Rv strain, a finding that supports the greater vulnerability of the former strain to T and to genetic depletion of QcrB. Against both strains, combinations of a DARQ, clofazimine (CFZ), and T were highly bactericidal. However, only against HN878 did T contribute synergistically, whereas an antagonistic effect was observed against H37Rv. These results demonstrate the therapeutic potential of T and highlight how differences in the susceptibility of M. tuberculosis strains could lead to different conclusions about a drugs potential contribution to novel drug regimens.

microbiology↗

bioRxiv2024

In-vitro resensitization of multidrug resistant clinical isolates of Enterococcus faecium and faecalis through phage-antibiotic synergy

Bacteriophages are an increasingly attractive option for the treatment of antibiotic resistant infections, but their efficacy is difficult to discern due to confounding effects of antibiotics. Phages are generally delivered in conjunction with antibiotics, and thus, when patients improve its unclear whether the phages, antibiotics or both are responsible. This question is particularly relevant for enterococcus infections, as limited data suggest phages might restore antibiotic efficacy against resistant strains. Enterococci can develop high-level resistance to vancomycin, a primary treatment. We assessed clinical and laboratory isolates of Enterococcus faecium and Enterococcus faecalis to determine whether we could observe synergistic interactions between phages and antibiotics. We identified synergy between multiple phages and antibiotics including linezolid, ampicillin, and vancomycin. Notably, antibiotic susceptibility did not predict synergistic interactions with phages. Vancomycin resistant isolates (n=6) were eradicated by the vancomycin-phage combination as effectively as vancomycin susceptible isolates (n=2). Transcriptome analysis revealed significant gene expression changes under antibiotic-phage conditions, especially for linezolid and vancomycin, with upregulated genes involved in nucleotide and protein biosynthesis and downregulated stress response and prophage-related genes. While our results do not conclusively determine the etiology of the observed synergistic interactions between antibiotics and phages, they do confirm and build upon previous research that observed these synergistic interactions. Our work highlights how using phages can restore the effectiveness of vancomycin against resistant isolates. This finding provides a promising, although unexpected, strategy for moving forward with phage treatments for Vancomycin Resistant Enterococcus infections.

microbiology↗

bioRxiv2024

Murine Progeria Model Exhibits Delayed Fracture Healing with Dysregulated Local Immune Response

ABSTRACTO_ST_ABSBackgroundC_ST_ABSBone fracture is one of the most globally prevalent injuries, with an estimated 189 million bone fractures occurring annually. Delayed union or nonunion occurs in up to 15% of fractures and involves the interruption or complete failure of bone continuity following fracture. Preclinical testing is essential to support the translation of novel strategies to promote improved fracture repair treatment, but there is a paucity of small animal models that recapitulate clinical attributes associated with delayed fracture healing. This study explores whether the Zmpste24-/- (Z24-/-) knockout mouse model of Hutchinson-Gilford progeria syndrome presents with delayed fracture healing. Leveraging the previously characterized Z24-/- phenotype of genomic instability, epigenetic changes, and fragility, we hypothesize that these underlying alterations will lead to significantly delayed fracture healing relative to age-matched wild type (WT) controls. MethodsWT and Z24-/- mice received intramedullary fixed tibia fractures at [~]12 weeks of age. Mice were sacrificed throughout the time course of repair for the collection of organs that would provide information regarding the local (fracture callus, bone marrow, inguinal lymph nodes) versus peripheral (peripheral blood, contralateral tibia, abdominal organs) tissue microenvironments. Analyses of these specimens include histomorphometry, CT, mechanical strength testing, protein quantification, gene expression analysis, flow cytometry for cellular senescence, and immunophenotyping. ResultsZ24-/- mice demonstrated a significantly delayed rate of healing compared to WT mice with consistently smaller fracture calli containing higher proportion of cartilage and less bone after injury. Cellular senescence and pro-inflammatory cytokines were elevated in the Z24-/- mice before and after fracture. These mice further presented with a dysregulated immune system, exhibiting generally decreased lymphopoiesis and increased myelopoiesis locally in the bone marrow, with more naive and less memory T cell but greater myeloid activation systemically in the peripheral blood. Surprisingly, the ipsilateral lymph nodes had increased T cell activation and other pro-inflammatory NK and myeloid cells, suggesting that elevated myeloid abundance and activation contributes to an injury-specific hyperactivation of T cells. ConclusionTaken together, these data establish the Z24-/- progeria mouse as a model of delayed fracture healing that exhibits decreased bone in the fracture callus, with weaker overall bone quality, immune dysregulation, and increased cellular senescence. Based on this mechanism for delayed healing, we propose this Z24-/- progeria mouse model could be useful in testing novel therapeutics that could address delayed healing. The Translational Potential of this ArticleThis study employs a novel animal model for delayed fracture healing that researchers can use to screen fracture healing therapeutics to address the globally prevalent issue of aberrant fracture healing.

molecular biology↗

bioRxiv2024

Pharmacological inhibition of macrophage triglyceride biosynthesis pathways does not improve Mycobacterium tuberculosis control in infected mice

Triglyceride rich macrophages (foam cells) are a hallmark of necrotic granulomas in tuberculosis, and multiple antimicrobial functions are down-regulated in these cells. In this study, we assessed the ability of two different compounds to reduce triglyceride content and intracellular burden in Mycobacterium tuberculosis (Mtb)-infected macrophages: A-922500 (DGATi), an inhibitor of diacylglycerol acyltransferase 1, an enzyme involved in triglyceride synthesis; and LY2584702 (p70S6Ki), an inhibitor of p70 S6 kinase, a serine/threonine kinase involved in mTORC-1dependent lipid biogenesis. Additionally, we evaluated the adjunctive activity of these inhibitors as host-directed therapies against chronic Mtb infection in C3HeB/FeJ mice. DGATi and p70S6Ki significantly reduced the lipid content and bacillary burden in Mtb-infected human monocyte-derived macrophages. In Mtb-infected mice, each inhibitor reduced the triglyceride content (P[≤] 0.0001) in cells from bronchoalveolar lavage samples. Adjunctive treatment of DGATi with isoniazid and p70S6Ki monotherapy reduced the lipid droplet content (P[≤] 0.05) within lung macrophages of Mtb-infected mice. However, neither inhibitor reduced the lung bacterial burden in Mtb-infected mice alone or in combination with isoniazid, and they did not alter lung inflammation. These findings provide further insights into the role of foam cells in tuberculosis pathogenesis and the utility of interventions targeting these cell populations as adjunctive host-directed therapies.

pharmacology and toxicology↗

bioRxiv2024

The Origins of ATP Dependence in Biological Nitrogen Fixation

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg2+, can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the key N2-reducing enzyme nitrogenase. We show that the ancestor has a strict requirement for ATP and its hydrolysis is coupled to electron transfer for N2 reduction. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme, and underscore how biomolecular constraints can entirely decouple cofactor selection from environmental availability.

evolutionary biology↗

bioRxiv2024

miR-203 controls timing of developmental transitions during early preimplantation embryogenesis

Commonly expressed at developmental transitions, microRNAs operate as fine tuners of gene expression to facilitate cell fate acquisition and lineage segregation. Nevertheless, how they might regulate the earliest developmental transitions in early mammalian embryogenesis remains obscure. Here, in a strictly in vivo approach based on novel genetically-engineered mouse models and single-cell RNA sequencing, we identify miR-203 as a critical regulator of timing and cell fate restriction within the totipotency to pluripotency transition in mouse embryos. Genetically engineered mouse models show that loss of miR-203 slows down developmental timing during preimplantation leading to the accumulation of embryos with high expression of totipotency-associated markers, including MERVL endogenous retroviral elements. A new embryonic reporter (eE-Reporter) transgenic mouse carrying MERVL-Tomato and Sox2-GFP transgenes showed that lack of miR-203 leads to sustained expression of MERVL and reduced Sox2 expression in preimplantation developmental stages. A combination of single-cell transcriptional studies and epigenetic analyses identified the central coactivator and histone acetyltransferase P300 as a major miR-203 target at the totipotency to pluripotency transition in vivo. By fine tuning P300 levels, miR-203 carves the epigenetic rewiring process needed for this developmental transition, allowing a timely and correctly paced development.

developmental biology↗