Oxidative stress is intrinsic to staphylococcal adaptation to fatty acid synthesis antibiotics.

Antibiotics inhibiting the fatty acid synthesis pathway (FASII) of the major pathogen Staphylococcus aureus reach their enzyme targets, but bacteria continue growth by using environmental fatty acids (eFAs) to produce phospholipids. We assessed the consequences and effectors of FASII-antibiotic (anti-FASII) adaptation. Anti-FASII induced lasting expression changes without genomic rearrangements. Several identified regulators affected the timing of adaptation outgrowth. Adaptation resulted in decreased expression of major virulence factors. Conversely, stress responses were globally increased and adapted bacteria were more resistant to peroxide killing. Importantly, pre-exposure to peroxide led to faster anti-FASII-adaptation by stimulating eFA incorporation. This adaptation differs from reports of peroxide-stimulated antibiotic efflux, which leads to tolerance. In vivo, anti-FASII-adapted S. aureus killed the insect host more slowly but continued multiplying. We conclude that staphylococcal adaptation to FASII antibiotics involves reprogramming, which decreases virulence and increases stress resistance. Peroxide, produced by the host to combat infection, favors anti-FASII adaptation.

Faecalibacterium duncaniae A2-165 regulates the expression of butyrate synthesis, ferrous iron uptake, and stress-response genes based on acetate consumption.

The promising next-generation probiotic Faecalibacterium prausnitzii is one of the most abundant acetate-consuming, butyrate-producing bacteria in the healthy human gut. Yet, little is known about how acetate availability affects this bacterium's gene expression strategies. Here, we investigated the effect of acetate on temporal changes in the transcriptome of F. duncaniae A2-165 cultures using RNA sequencing. We compared gene expression patterns between two growth phases (early stationary vs. late exponential) and two acetate levels (low: 3 mM vs. high: 23 mM). Only in low-acetate conditions, a general stress response was activated. In high-acetate conditions, there was greater expression of genes related to butyrate synthesis and to the importation of B vitamins and iron. Specifically, expression was strongly activated in the case of the feoAABC operon, which encodes a FeoB ferrous iron transporter, but not in the case of the feoAB gene, which encodes a second putative FeoAB transporter. Moreover, excess ferrous iron repressed feoB expression but not feoAB. Lastly, FeoB but not FeoAB peptides from strain A2-165 were found in abundance in a healthy human fecal metaproteome. In conclusion, we characterized two early-stationary transcriptomes based on acetate consumption and this work highlights the regulation of feoB expression in F. duncaniae A2-165.

Specific Milk Composition of miR-30b Transgenic Mice Associated with Early Duodenum Maturation in Offspring with Lasting Consequences for Growth.

BACKGROUND: Milk composition is complex and includes numerous components essential for offspring growth and development. In addition to the high abundance of miR-30b microRNA, milk produced by the transgenic mouse model of miR-30b-mammary deregulation displays a significantly altered fatty acid profile. Moreover, wild-type adopted pups fed miR-30b milk present an early growth defect. OBJECTIVE: This study aimed to investigate the consequences of miR-30b milk feeding on the duodenal development of wild-type neonates, a prime target of suckled milk, along with comprehensive milk phenotyping. METHODS: The duodenums of wild-type pups fed miR-30b milk were extensively characterized at postnatal day (PND)-5, PND-6, and PND-15 using histological, transcriptomic, proteomic, and duodenal permeability analyses and compared with those of pups fed wild-type milk. Milk of miR-30b foster dams collected at mid-lactation was extensively analyzed using proteomic, metabolomic, and lipidomic approaches and hormonal immunoassays. RESULTS: At PND-5, wild-type pups fed miR-30b milk showed maturation of their duodenum with 1.5-fold (P < 0.05) and 1.3-fold (P < 0.10) increased expression of Claudin-3 and Claudin-4, respectively, and changes in 8 duodenal proteins (P < 0.10), with an earlier reduction in paracellular and transcellular permeability (183 ng/mL fluorescein sulfonic acid [FSA] and 12 ng/mL horseradish peroxidase [HRP], respectively, compared with 5700 ng/mL FSA and 90 ng/mL HRP in wild-type; P < 0.001). Compared with wild-type milk, miR-30b milk displayed an increase in total lipid (219 g/L compared with 151 g/L; P < 0.05), ceramide (17.6 µM compared with 6.9 µM; P < 0.05), and sphingomyelin concentrations (163.7 µM compared with 76.3 µM; P < 0.05); overexpression of 9 proteins involved in the gut barrier (P < 0.1); and higher insulin and leptin concentrations (1.88 ng/mL and 2.04 ng/mL, respectively, compared with 0.79 ng/mL and 1.06 ng/mL; P < 0.01). CONCLUSIONS: miR-30b milk displays significant changes in bioactive components associated with neonatal duodenal integrity and maturation, which could be involved in the earlier intestinal closure phenotype of the wild-type pups associated with a lower growth rate.

Wood Degradation by Fomitiporia mediterranea M. Fischer: Exploring Fungal Adaptation Using Metabolomic Networking.

Fomitiporia mediterranea M. Fischer (Fmed) is a white-rot wood-decaying fungus associated with one of the most important and challenging diseases in vineyards: Esca. To relieve microbial degradation, woody plants, including Vitis vinifera, use structural and chemical weapons. Lignin is the most recalcitrant of the wood cell wall structural compounds and contributes to wood durability. Extractives are constitutive or de novo synthesized specialized metabolites that are not covalently bound to wood cell walls and are often associated with antimicrobial properties. Fmed is able to mineralize lignin and detoxify toxic wood extractives, thanks to enzymes such as laccases and peroxidases. Grapevine wood's chemical composition could be involved in Fmed's adaptation to its substrate. This study aimed at deciphering if Fmed uses specific mechanisms to degrade grapevine wood structure and extractives. Three different wood species, grapevine, beech, and oak. were exposed to fungal degradation by two Fmed strains. The well-studied white-rot fungus Trametes versicolor (Tver) was used as a comparison model. A simultaneous degradation pattern was shown for Fmed in the three degraded wood species. Wood mass loss after 7 months for the two fungal species was the highest with low-density oak wood. For the latter wood species, radical differences in initial wood density were observed. No differences between grapevine or beech wood degradation rates were observed after degradation by Fmed or by Tver. Contrary to the Tver secretome, one manganese peroxidase isoform (MnP2l, jgi protein ID 145801) was the most abundant in the Fmed secretome on grapevine wood only. Non-targeted metabolomic analysis was conducted on wood and mycelium samples, using metabolomic networking and public databases (GNPS, MS-DIAL) for metabolite annotations. Chemical differences between non-degraded and degraded woods, and between mycelia grown on different wood species, are discussed. This study highlights Fmed physiological, proteomic and metabolomic traits during wood degradation and thus contributes to a better understanding of its wood degradation mechanisms.

A proteome scale study reveals how plastic surfaces and agitation promote protein aggregation.

Protein aggregation in biotherapeutics can reduce their activity and effectiveness. It may also promote immune reactions responsible for severe adverse effects. The impact of plastic materials on protein destabilization is not totally understood. Here, we propose to deconvolve the effects of material surface, air/liquid interface, and agitation to decipher their respective role in protein destabilization and aggregation. We analyzed the effect of polypropylene, TEFLON, glass and LOBIND surfaces on the stability of purified proteins (bovine serum albumin, hemoglobin and α-synuclein) and on a cell extract composed of 6000 soluble proteins during agitation (P = 0.1-1.2 W/kg). Proteomic analysis revealed that chaperonins, intrinsically disordered proteins and ribosomes were more sensitive to the combined effects of material surfaces and agitation while small metabolic oligomers could be protected in the same conditions. Protein loss observations coupled to Raman microscopy, dynamic light scattering and proteomic allowed us to propose a mechanistic model of protein destabilization by plastics. Our results suggest that protein loss is not primarily due to the nucleation of small aggregates in solution, but to the destabilization of proteins exposed to material surfaces and their subsequent aggregation at the sheared air/liquid interface, an effect that cannot be prevented by using LOBIND tubes. A guidance can be established on how to minimize these adverse effects. Remove one of the components of this combined stress - material, air (even partially), or agitation - and proteins will be preserved.

Peripheral proteomic changes after electroconvulsive seizures in a rodent model of non-response to chronic fluoxetine.

Major depressive disorder (MDD) is the psychiatric disorder with the highest prevalence in the world. Pharmacological antidepressant treatment (AD), such as selective serotonin reuptake inhibitors [SSRI, i.e., fluoxetine (Flx)] is the first line of treatment for MDD. Despite its efficacy, lack of AD response occurs in numerous patients characterizing Difficult-to-treat Depression. ElectroConvulsive Therapy (ECT) is a highly effective treatment inducing rapid improvement in depressive symptoms and high remission rates of ∼50-63% in patients with pharmaco-resistant depression. Nevertheless, the need to develop reliable treatment response predictors to guide personalized AD strategies and supplement clinical observation is becoming a pressing clinical objective. Here, we propose to establish a proteomic peripheral biomarkers signature of ECT response in an anxio/depressive animal model of non-response to AD. Using an emotionality score based on the analysis complementary behavioral tests of anxiety/depression (Elevated Plus Maze, Novelty Suppressed Feeding, Splash Test), we showed that a 4-week corticosterone treatment (35 µg/ml, Cort model) in C57BL/6JRj male mice induced an anxiety/depressive-like behavior. A 28-day chronic fluoxetine treatment (Flx, 18 mg/kg/day) reduced corticosterone-induced increase in emotional behavior. A 50% decrease in emotionality score threshold before and after Flx, was used to separate Flx-responding mice (Flx-R, n = 18), or Flx non-responder mice (Flx-NR, n = 7). Then, Flx-NR mice received seven sessions of electroconvulsive seizure (ECS, equivalent to ECT in humans) and blood was collected before and after ECS treatment. Chronic ECS normalized the elevated emotionality observed in Flx-NR mice. Then, proteins were extracted from peripheral blood mononuclear cells (PBMCs) and isolated for proteomic analysis using a high-resolution MS Orbitrap. Data are available via ProteomeXchange with identifier PXD037392. The proteomic analysis revealed a signature of 33 peripheral proteins associated with response to ECS (7 down and 26 upregulated). These proteins were previously associated with mental disorders and involved in regulating pathways which participate to the depressive disorder etiology.

A unique, newly discovered four-member protein family involved in extracellular fatty acid binding in Yarrowia lipolytica.

BACKGROUND: Yarrowia lipolytica, a nonconventional oleaginous yeast species, has attracted attention due to its high lipid degradation and accumulation capacities. Y. lipolytica is used as a chassis for the production of usual and unusual lipids and lipid derivatives. While the genes involved in the intracellular transport and activation of fatty acids in different cellular compartments have been characterized, no genes involved in fatty acid transport from the extracellular medium into the cell have been identified thus far. In this study, we identified secreted proteins involved in extracellular fatty acid binding. RESULTS: Recent analysis of the Y. lipolytica secretome led to the identification of a multigene family that encodes four secreted proteins, preliminarily named UP1 to UP4. These proteins were efficiently overexpressed individually in wild-type and multideletant strain (Q4: Δup1Δup2Δup3Δup4) backgrounds. Phenotypic analysis demonstrated the involvement of these proteins in the binding of extracellular fatty acids. Additionally, gene deletion and overexpression prevented and promoted sensitivity to octanoic acid (C8) toxicity, respectively. The results suggested binding is dependent on aliphatic chain length and fatty acid concentration. 3D structure modeling supports the proteins' role in fatty acid assimilation at the molecular level. CONCLUSIONS: We discovered a family of extracellular-fatty-acid-binding proteins in Y. lipolytica and have proposed to name its members eFbp1 to eFbp4. The exact mode of eFbps action remains to be deciphered individually and synergistically; nevertheless, it is expected that the proteins will have applications in lipid biotechnology, such as improving fatty acid production and/or bioconversion.

Top-Down proteomics based on LC-MS combined with cDNA sequencing to characterize multiple proteoforms of Amiata donkey milk proteins.

An in-depth molecular characterization of the main milk proteins, caseins (CNs) and whey proteins, from Amiata donkey combining top-down proteomic analysis (LC-MS) and cDNA sequencing revealed multiple proteoforms arising from complex splicing patterns, including cryptic splice site usage and exon skipping events. Post-translational modifications, in particular phosphorylation, increased the variety and complexity of proteoforms. αs2-CN perfectly exemplifies such a complexity. With 2 functional genes, CSN1S2 I and CSN1S2 II, made of 20 and 16 exons respectively, nearly 30 different molecules of this CN were detected in the milk of one Amiata donkey. A cryptic splice site usage, leading to a singular shift of the open reading frame and generating two αs2-CN I isoforms with different C-terminal sequences, was brought to light. Twenty different αs1-CN molecules with different phosphorylation levels ranging between 4 and 9P were identified in a single milk sample, most of them resulting from exon skipping events and cryptic splice site usage. Novel genetic polymorphisms were detected for CNs (ß- and αs-CN) as well as for whey proteins (lysozyme C and ß-LG I). The probable new ß-LG I variant, with a significantly higher mass than known variants, appears to display an N-terminal extension possibly related to the signal peptide sequence. This represents the most comprehensive report to date detailing the complexity of donkey milk protein micro-heterogeneity, a prerequisite for discovering new elements to objectify the original properties of donkey's milk.

Corrigendum: An Immunomodulatory Transcriptional Signature Associated With Persistent Listeria Infection in Hepatocytes.

[This corrects the article DOI: 10.3389/fcimb.2021.761945.].

Reconstruction of a catalogue of genome-scale metabolic models with enzymatic constraints using GECKO 2.0.

Genome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into such models was first enabled by the GECKO toolbox, allowing the study of phenotypes constrained by protein limitations. Here, we upgrade the toolbox in order to enhance models with enzyme and proteomics constraints for any organism with a compatible GEM reconstruction. With this, enzyme-constrained models for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus are generated to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions reveal that upregulation and high saturation of enzymes in amino acid metabolism are common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO is expanded with an automated framework for continuous and version-controlled update of enzyme-constrained GEMs, also producing such models for Escherichia coli and Homo sapiens. In this work, we facilitate the utilization of enzyme-constrained GEMs in basic science, metabolic engineering and synthetic biology purposes.

Modern Metaproteomics: A Unique Tool to Characterize the Active Microbiome in Health and Diseases, and Pave the Road towards New Biomarkers-Example of Crohn's Disease and Ulcerative Colitis Flare-Ups.

Thanks to the latest developments in mass spectrometry, software and standards, metaproteomics is emerging as the vital complement of metagenomics, to make headway in understanding the actual functioning of living and active microbial communities. Modern metaproteomics offers new possibilities in the area of clinical diagnosis. This is illustrated here, for the still highly challenging diagnosis of intestinal bowel diseases (IBDs). Using bottom-up proteomics, we analyzed the gut metaproteomes of the same twenty faecal specimens processed either fresh or after a two-month freezing period. We focused on metaproteomes of microbial cell envelopes since it is an outstanding way of capturing host and host-microbe interaction signals. The protein profiles of pairs of fresh and frozen-thawed samples were closely related, making feasible deferred analysis in a distant diagnosis centre. The taxonomic and functional landscape of microbes in diverse IBD phenotypes-active ulcerative colitis, or active Crohn's disease either with ileo-colonic or exclusive colonic localization-differed from each other and from the controls. Based on their specific peptides, we could identify proteins that were either strictly overrepresented or underrepresented in all samples of one clinical group compared to all samples of another group, paving the road for promising additional diagnostic tool for IBDs.

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures.

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

Comparison of Different Label-Free Techniques for the Semi-Absolute Quantification of Protein Abundance.

In proteomics, it is essential to quantify proteins in absolute terms if we wish to compare results among studies and integrate high-throughput biological data into genome-scale metabolic models. While labeling target peptides with stable isotopes allow protein abundance to be accurately quantified, the utility of this technique is constrained by the low number of quantifiable proteins that it yields. Recently, label-free shotgun proteomics has become the "gold standard" for carrying out global assessments of biological samples containing thousands of proteins. However, this tool must be further improved if we wish to accurately quantify absolute levels of proteins. Here, we used different label-free quantification techniques to estimate absolute protein abundance in the model yeast Saccharomyces cerevisiae. More specifically, we evaluated the performance of seven different quantification methods, based either on spectral counting (SC) or extracted-ion chromatogram (XIC), which were applied to samples from five different proteome backgrounds. We also compared the accuracy and reproducibility of two strategies for transforming relative abundance into absolute abundance: a UPS2-based strategy and the total protein approach (TPA). This study mentions technical challenges related to UPS2 use and proposes ways of addressing them, including utilizing a smaller, more highly optimized amount of UPS2. Overall, three SC-based methods (PAI, SAF, and NSAF) yielded the best results because they struck a good balance between experimental performance and protein quantification.

The Metaproteomics Initiative: a coordinated approach for propelling the functional characterization of microbiomes.

Through connecting genomic and metabolic information, metaproteomics is an essential approach for understanding how microbiomes function in space and time. The international metaproteomics community is delighted to announce the launch of the Metaproteomics Initiative (www.metaproteomics.org), the goal of which is to promote dissemination of metaproteomics fundamentals, advancements, and applications through collaborative networking in microbiome research. The Initiative aims to be the central information hub and open meeting place where newcomers and experts interact to communicate, standardize, and accelerate experimental and bioinformatic methodologies in this field. We invite the entire microbiome community to join and discuss potential synergies at the interfaces with other disciplines, and to collectively promote innovative approaches to gain deeper insights into microbiome functions and dynamics. Video Abstract.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

An Immunomodulatory Transcriptional Signature Associated With Persistent Listeria Infection in Hepatocytes.

Listeria monocytogenes causes severe foodborne illness in pregnant women and immunocompromised individuals. After the intestinal phase of infection, the liver plays a central role in the clearance of this pathogen through its important functions in immunity. However, recent evidence suggests that during long-term infection of hepatocytes, a subpopulation of Listeria may escape eradication by entering a persistence phase in intracellular vacuoles. Here, we examine whether this long-term infection alters hepatocyte defense pathways, which may be instrumental for bacterial persistence. We first optimized cell models of persistent infection in human hepatocyte cell lines HepG2 and Huh7 and primary mouse hepatocytes (PMH). In these cells, Listeria efficiently entered the persistence phase after three days of infection, while inducing a potent interferon response, of type I in PMH and type III in HepG2, while Huh7 remained unresponsive. RNA-sequencing analysis identified a common signature of long-term Listeria infection characterized by the overexpression of a set of genes involved in antiviral immunity and the under-expression of many acute phase protein (APP) genes, particularly involved in the complement and coagulation systems. Infection also altered the expression of cholesterol metabolism-associated genes in HepG2 and Huh7 cells. The decrease in APP transcripts was correlated with lower protein abundance in the secretome of infected cells, as shown by proteomics, and also occurred in the presence of APP inducers (IL-6 or IL-1ß). Collectively, these results reveal that long-term infection with Listeria profoundly deregulates the innate immune functions of hepatocytes, which could generate an environment favorable to the establishment of persistent infection.

Mechanisms of Acetoin Toxicity and Adaptive Responses in an Acetoin-Producing Species, Lactococcus lactis.

Acetoin, 3-hydroxyl,2-butanone, is extensively used as a flavor additive in food products. This volatile compound is produced by the dairy bacterium Lactococcus lactis when aerobic respiration is activated by haem addition, and comprises ∼70% of carbohydrate degradation products. Here we investigate the targets of acetoin toxicity, and determine how acetoin impacts L. lactis physiology and survival. Acetoin caused damage to DNA and proteins, which related to reactivity of its keto group. Acetoin stress was reflected in proteome profiles, which revealed changes in lipid metabolic proteins. Acetoin provoked marked changes in fatty acid composition, with massive accumulation of cycC19:0 cyclopropane fatty acid at the expense of its unsaturated C18:1 fatty acid precursor. Deletion of the cfa gene, encoding the cycC19:0 synthase, sensitized cells to acetoin stress. Acetoin-resistant transposon mutagenesis revealed a hot spot in the high affinity phosphate transporter operon pstABCDEF, which is known to increase resistance to multiple stresses. This work reveals the causes and consequences of acetoin stress on L. lactis, and may facilitate control of lactic acid bacteria production in technological processes. IMPORTANCE Acetoin, 3-hydroxyl,2-butanone, has diverse uses in chemical industry, agriculture, and dairy industries as a volatile compound that generates aromas. In bacteria, it can be produced in high amount by Lactococcus lactis when it grows under aerobic respiration. However, acetoin production can be toxic and detrimental for growth and/or survival. Our results showed that it damages DNA and proteins via its keto group. We also showed that acetoin modifies membrane fatty acid composition with the production of cyclopropane C19:0 fatty acid at the expense of an unsaturated C18:1. We isolated mutants more resistant to acetoin than the wild-type strain. All of them mapped to a single locus pstABCDEF operon, suggesting a simple means to limit acetoin toxicity in dairy bacteria and to improve its production.

Coelimycin Synthesis Activatory Proteins Are Key Regulators of Specialized Metabolism and Precursor Flux in Streptomyces coelicolor A3(2).

Many microbial specialized metabolites are industrially relevant agents but also serve as signaling molecules in intra-species and even inter-kingdom interactions. In the antibiotic-producing Streptomyces, members of the SARP (Streptomyces antibiotic regulatory proteins) family of regulators are often encoded within biosynthetic gene clusters and serve as their direct activators. Coelimycin is the earliest, colored specialized metabolite synthesized in the life cycle of the model organism Streptomyces coelicolor A3(2). Deletion of its two SARP activators cpkO and cpkN abolished coelimycin synthesis and resulted in dramatic changes in the production of the later, stationary-phase antibiotics. The underlying mechanisms of these phenotypes were deregulation of precursor flux and quorum sensing, as shown by label-free, bottom-up shotgun proteomics. Detailed profiling of promoter activities demonstrated that CpkO is the upper-level cluster activator that induces CpkN, while CpkN activates type II thioesterase ScoT, necessary for coelimycin synthesis. What is more, we show that cpkN is regulated by quorum sensing gamma-butyrolactone receptor ScbR.

Cardiolipin content controls mitochondrial coupling and energetic efficiency in muscle.

Unbalanced energy partitioning participates in the rise of obesity, a major public health concern in many countries. Increasing basal energy expenditure has been proposed as a strategy to fight obesity yet raises efficiency and safety concerns. Here, we show that mice deficient for a muscle-specific enzyme of very-long-chain fatty acid synthesis display increased basal energy expenditure and protection against high-fat diet-induced obesity. Mechanistically, muscle-specific modulation of the very-long-chain fatty acid pathway was associated with a reduced content of the inner mitochondrial membrane phospholipid cardiolipin and a blunted coupling efficiency between the respiratory chain and adenosine 5'-triphosphate (ATP) synthase, which was restored by cardiolipin enrichment. Our study reveals that selective increase of lipid oxidative capacities in skeletal muscle, through the cardiolipin-dependent lowering of mitochondrial ATP production, provides an effective option against obesity at the whole-body level.

Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick-Pathogen Interactions.

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick-pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick-pathogen interactions.