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Salicylic acid, as a plant hormone, significantly affects the physiological and biochemical indexes of soluble sugar, malondialdehyde content, peroxidase, and superoxide dismutase enzyme activity in Platycodon grandiflorus. Lysine malonylation is a post-translational modification that involves various cellular functions in plants, though it is rarely studied, especially in medicinal plants. In this study, the aim was to perform a comparative quantitative proteomic study of malonylation modification on P. grandiflorus root proteins after salicylic acid treatment using Western blot with specific antibodies, affinity enrichment and LC-MS/MS analysis methods. The analysis identified 1907 malonyl sites for 809 proteins, with 414 proteins and 798 modification sites quantified with high confidence. Post-treatment, 361 proteins were upregulated, and 310 were downregulated. Bioinformatics analysis revealed that malonylation in P. grandiflorus is primarily involved in photosynthesis and carbon metabolism. Physiological and biochemical analysis showed that salicylic acid treatment increased the malondialdehyde levels, soluble protein, superoxide dismutase, and peroxidase activity but did not significantly affect the total saponins content in P. grandiflorus. These findings provide an important basis for exploring the molecular mechanisms of P. grandiflorus following salicylic acid treatment and enhance understanding of the biological function of protein lysine malonylation in plants.

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Late onset Pompe disease (LOPD) is caused by a deficiency of the enzyme acid α-glucosidase, resulting in glycogen accumulation in lysosomes. The mechanism of LOPD has been less explored. In this study, we used an integrative analysis of the proteomics and metabolomics of LOPD muscle samples to reveal the potential mechanisms. Proteomic analysis identified 635 upregulated proteins and 89 downregulated proteins in the LOPD group. Similarly, metabolomic analysis revealed 15 upregulated and 143 downregulated metabolites; notably, L-arginine levels were significantly decreased in the LOPD group. Lysosome-related GO terms were significantly upregulated, while GO terms related to neurofilament, cytoskeleton, axon ensheathment, and myelin sheath were significantly downregulated. KEGG pathway analysis demonstrated that the lysosome, autophagy, and mTOR pathways were distinctly upregulated. Correlation analysis indicated that CALML3 showed a potential correlation with LOPD severity. Our study highlighted the potential crosstalk among these LOPD-related pathways. Supplementation with L-arginine could represent a promising therapeutic approach for LOPD, and CALML3 could serve as a potential biomarker for LOPD severity. These findings provide valuable insights into the pathogenesis of LOPD and suggest avenues for future therapeutic development.

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As complex quantitative traits, soybean seed oil and protein contents are governed by dynamic proteome networks that remain largely unknown. Here, we investigated the dynamic changes of the proteome during seed maturation across two soybean varieties with contrasting seed oil and protein content. Through optimizing the detectability of low-abundance proteins and utilizing library-free data-independent acquisition (directDIA) mass spectrometry, we unprecedentedly identified 7414 proteins and 3975 protein groups (PGs), substantially expanding the soybean seed proteome. Among the PGs, 1391 differentially accumulated between the two varieties. By comparing the abundance of PGs between the two varieties, we identified the core and periphery proteome of soybean seeds and revealed that variations in the oil and protein content are primarily attributed to the peripheral proteome, which significantly fluctuated across seed developmental stages. Our work presents a quantitative proteomic atlas underlying the variation of seed oil and protein content in soybean varieties and provides insight into the mechanisms regulating the seed oil and protein content in soybean.

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In recent years, it was found that lysine malonylation modification can affect biological metabolism and play an important role in plant life activities. Platycodon grandiflorus, an economic crop and medicinal plant, had no reports on malonylation in the related literature. This study qualitatively introduces lysine malonylation in P. grandiflorus. A total of 888 lysine malonylation-modified proteins in P. grandiflorus were identified, with a total of 1755 modification sites. According to the functional annotation, malonylated proteins were closely related to catalysis, binding, and other reactions. Subcellular localization showed that related proteins were enriched in chloroplasts, cytoplasm, and nuclei, indicating that this modification could regulate various metabolic processes. Motif analysis showed the enrichment of Alanine (A), Cysteine (C), Glycine (G), and Valine (V) amino acids surrounding malonylated lysine residues. Metabolic pathway and protein-protein interaction network analyses suggested these modifications are mainly involved in plant photosynthesis. Moreover, malonylated proteins are also involved in stress and defense responses. This study shows that lysine malonylation can affect a variety of biological processes and metabolic pathways, and the contents are reported for the first time in P. grandiflorus, which can provide important information for further research on P. grandiflorus and lysine malonylation's role in environment stress, photosynthesis, and secondary metabolites enrichment.

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It is well known that individual pea (Pisum sativum L.) cultivars differ in their symbiotic responsivity. This trait is typically manifested with an increase in seed weights, due to inoculation with rhizobial bacteria and arbuscular mycorrhizal fungi. The aim of this study was to characterize alterations in the root proteome of highly responsive pea genotype k-8274 plants and low responsive genotype k-3358 ones grown in non-sterile soil, which were associated with root colonization with rhizobial bacteria and arbuscular mycorrhizal fungi (in comparison to proteome shifts caused by soil supplementation with mineral nitrogen salts). Our results clearly indicate that supplementation of the soil with mineral nitrogen-containing salts switched the root proteome of both genotypes to assimilation of the available nitrogen, whereas the processes associated with nitrogen fixation were suppressed. Surprisingly, inoculation with rhizobial bacteria had only a minor effect on the root proteomes of both genotypes. The most pronounced response was observed for the highly responsive k-8274 genotype inoculated simultaneously with rhizobial bacteria and arbuscular mycorrhizal fungi. This response involved activation of the proteins related to redox metabolism and suppression of excessive nodule formation. In turn, the low responsive genotype k-3358 demonstrated a pronounced inoculation-induced suppression of protein metabolism and enhanced diverse defense reactions in pea roots under the same soil conditions. The results of the study shed light on the molecular basis of differential symbiotic responsivity in different pea cultivars. The raw data are available in the PRIDE repository under the project accession number PXD058701 and project DOI 10.6019/PXD058701.

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Heat stress can disrupt the balance between the heat poultry release into the environment and the heat they generate. Pequi oil has antioxidant properties, which may mitigate the heat stress effects. This study aimed to investigate the response of laying hens to pequi oil supplementation under heat stress using a proteomic approach. A total of 96 Lohmann White laying hens with 26 weeks old were housed in a completely randomized design with a 2 × 2 factorial arrangement. They were housed in two climate chambers, thermal comfort temperature ± 24.04 °C with the relative humidity ± 66.35 and heat stress (HS) ± 31.26 °C with the relative humidity ± 60.62. They were fed two diets: a control diet (CON), basal diet (BD) without additives, and with Pequi oil (PO), BD + 0.6% PO. After 84 days, plasma samples were analyzed using Shotgun and LC-MS/MS. Proteins related to anti-inflammation, transport, and the immune system were differentially expressed in hens fed PO and CON under heat stress compared to those in thermoneutral environments. This helps protect against oxidative stress and may support the body's ability to manage heat-induced damage, stabilizing protein expression under stress conditions. The ovotransferrin proteins, fibrinogen isoforms, apolipoprotein A-I, Proteasome activator subunit 4, Transthyretin, and the enzyme serine Peptidase Inhibitor_Kazal Type 5, which presented Upregulated (Up) equal to 1, present characteristics that may be crucial for enhancing the adaptive responses of hens to thermal stress, thereby increasing their tolerance and minimizing the negative effects of heat on egg production. The data presented in this manuscript provides new insights into the plasma proteome alterations of laying hens fed a diet supplemented with pequi oil during heat stress challenges.

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Results of artificial insemination (AI) are affected by changes in sperm quality and the function throughout collection and preservation procedures. Proteome and metabolome alterations of sperm treated with the different procedures in goat, however, aren't fully understood. To this end, we sought to investigate the impacts of rectal probe electrostimulation (EE) and artificial vagina (AV) semen collection methods on the quality and the cryotolerance of goat sperm, with additional focus on proteomic and metabolomic analyses. Semen samples were collected from Yunshang black goats and categorized into four groups: fresh sperm collected via AV (XAZ), fresh sperm collected via EE (XEZ), frozen sperm post-AV collection (DAZ) and frozen sperm post-EE collection (DEZ). Four comparisons (XAZ vs. XEZ, DAZ vs. XAZ, DEZ vs. XEZ, DAZ vs. DEZ) were performed, respectively. This study first evaluated sperm motility, acrosome integrity, plasma membrane integrity, mitochondrial activity, and reactive oxygen species (ROS) levels. The results indicated that there were no significant differences in fresh sperm quality parameters between the EE and AV methods. However, notable differences emerged post-cryopreservation. Specifically, the AV method proved more advantageous in preserving the motility, integrities of acrosome and plasma membrane, mitochondrial activity of frozen sperm compared to the EE method. Through the multi-omics approaches, a total of 210 differentially abundant proteins (DAPs) related to sperm characteristics and function were identified across the four comparations. Moreover, 32 differentially abundant metabolites (DAMs) were detected. Comprehensive bioinformatics analysis underscored significant molecular pathways in the co-enrichment of DAPs and DAMs, particularly focusing on the citrate cycle, ROS, oxidative phosphorylation, and glycine, serine, and threonine metabolism etc. We elucidated the differential impacts of AV and EE collection methods on the quality and cryotolerance of goat semen from omics perspectives, which offer a critical foundation for further exploration into optimizing semen collection and cryopreservation techniques in goat breeding program.

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In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to approximate compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified more than one million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.

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Sulfur is an essential nutrient for various physiological processes, including protein synthesis and enzyme activation. We aimed to evaluate how S-benzyl-L-cysteine (SBC), an inhibitor of the sulfur assimilation pathway, affects maize plants' growth, photosynthesis, and leaf proteomic profile. Thus, maize plants were grown for 14 days in vermiculite supplemented with SBC. Photosynthesis was assessed using light and CO2 response curves and chlorophyll a fluorescence. Leaf proteome analysis was conducted to evaluate photosynthetic protein biosynthesis, and ROS content was quantified to assess oxidative stress. Applying SBC resulted in a significant decrease in the growth of maize plants. The gas exchange analysis revealed that maize plants exhibited a diminished rate of CO2 assimilation attributable to both stomatal and non-stomatal limitations. Furthermore, SBC suppressed the activity of important elements involved in the photosynthetic electron transport chain (including photosystems I and II, cytochrome b6f, and ATP synthase) and enzymes responsible for the Calvin cycle, some of which have sulfur-containing prosthetic groups. Consequently, the diminished electron flow rate resulted in a substantial increase in the levels of ROS within the leaves. Our research highlights the crucial role of SBC in disrupting maize photosynthesis by limiting L-cysteine and assimilated sulfur availability, which are essential for the synthesis of protein and prosthetic groups and photosynthetic processes, emphasizing the potential of OAS-TL as a new herbicide site of action.

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Xylanibacter ruminicola is an abundant rumen bacterium that produces propionate in a cobalamin (vitamin B12)-dependent manner via the succinate pathway. However, the extent to which this occurs across ruminal Xylanibacter and closely related bacteria, and the effect of cobalamin supplementation on the expression of propionate pathway genes and enzymes has yet to be investigated. To assess this, we screened 14 strains and found that almost all strains produced propionate when supplemented with cobalamin. X. ruminicola KHP1 was selected for further study, including complete genome sequencing, and comparative transcriptomics and proteomics of KHP1 cultures grown with and without supplemented cobalamin. The complete KHP1 genome was searched for cobalamin-binding riboswitches and four were predicted, though none were closely located to any of the succinate pathway genes, which were dispersed at numerous genomic loci. Cobalamin supplementation led to the differential expression of 17.5% of genes, including genes encoding the cobalamin-dependent methylmalonyl-CoA mutase and some methylmalonyl-CoA decarboxylase subunits, but most propionate biosynthesis pathway genes were not differentially expressed. The effect of cobalamin supplementation on the KHP1 proteome was much less pronounced, with the only differentially abundant propionate pathway enzyme being methylmalonyl-CoA mutase, which had greater abundance when supplemented with cobalamin. Our results demonstrate that cobalamin supplementation does not result in induction of the entire propionate biosynthesis pathway, but consistently increased expression of methylmalonyl-CoA mutase at transcriptome and proteome levels. The magnitude of the differential expression of propionate pathway genes observed was minor compared to that of genes proximate to predicted cobalamin riboswitches. IMPORTANCE: In ruminants, the rumen microbial community plays a critical role in nutrition through the fermentation of feed to provide vital energy substrates for the host animal. Propionate is a major rumen fermentation end-product and increasing its production is desirable given its importance in host glucose production and impact on greenhouse gas production. Vitamin B12 (cobalamin) can induce propionate production in the prominent rumen bacterium Xylanibacter ruminicola, but it is not fully understood how cobalamin regulates propionate pathway activity. Contrary to expectation, we found that cobalamin supplementation had little effect on propionate pathway expression at transcriptome and proteome levels, with minor upregulation of genes encoding the cobalamin-dependent enzyme of the pathway. These findings provide new insights into factors that regulate propionate production and suggest that cobalamin-dependent propionate production by X. ruminicola is controlled post-translationally.

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The impact of nutritional modification to increase functional polyunsaturated fatty acids (PUFA), such as n-3 and n-6 fatty acids (FA) or conjugated linoleic acid (CLA), on milk proteome profile during early lactation remains largely unknown. We used an untargeted proteomics approach to investigate the impact of lactation day and PUFA supplementation on the proteome signature in skimmed milk over the course of early lactation. Sixteen Holstein dairy cows received abomasal infusion of saturated FA (CTRL) or a mixture of essential FA and CLA (EFA + CLA group) from - 63 to + 63 days relative to parturition. Using quantitative proteomics, 479 unique proteins were identified in skimmed milk at days 1, 28, and 63 postpartum. The top discriminating proteins between transition milk (day 1) and mature milk (days 28 and 63), including members of complements (i.e. C2 and C5), growth factor (TGFB2), lipoproteins (i.e. APOE and APOD), and chaperones (i.e. ST13 and CLU), are associated with calves' immune system and gut development. The EFA + CLA supplementation moderately affected a few proteins associated with regulating mammary glands' lipogenesis through the (re)assembly of lipoprotein particles, possibly under the PPAR signaling pathway. Collectively, skimmed milk proteome is dynamically regulated initially by cow's metabolic and physiological changes and to a lesser extent by nutritional PUFA modifications.

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BACKGROUND: Cormus domestica (L.) is a monophyletic wild fruit tree belonging to the Rosaceae family, with well-documented use in the Mediterranean region. Traditionally, these fruits are harvested and stored for at least 2 weeks before consumption. During this period, the fruit reaches its well-known and peculiar organoleptic and texture characteristics. However, the spread of more profitable fruit tree species, resulted in its progressive erosion. In this work we performed proteomic and metabolomic fruit analyses at three times after harvesting, to characterise postharvest physiological and molecular changes, it related to nutritional and organoleptic properties at consumption. RESULTS: Proteomics and metabolomics analysis were performed on fruits harvested at different time points: freshly harvested fruit (T0), fruit two weeks after harvest (T1) and fruit four weeks after harvest (T2). Proteomic analysis (Shotgun Proteomic in LC-MS/MS) resulted in 643 proteins identified. Most of the differentially abundant proteins between the three phases observed were involved in the softening process, carbohydrate metabolism and stress responses. Enzymes, such as xyloglucan endotransglucosylase/hydrolase, pectin acetylesterase, beta-galactosidase and pectinesterase, accumulated during fruit ripening and could explain the pulp breakdown observed in C. domestica. At the same time, enzymes abundant in the early stages (T0), such as sucrose synthase and malic enzyme, explain the accumulation of sugars and the lowering of acidity during the process. The metabolites extraction from C. domestica fruits enabled the identification of 606 statistically significant differentially abundant metabolites. Some compounds such as piptamine and resorcinol, well-known for their antimicrobial and antifungal properties, and several bioactive compounds such as endocannabinoids, usually described in the leaves, accumulate in C. domestica fruit during the post-harvest process. CONCLUSIONS: The metabolomic and proteomic profiling of the C. domestica fruit during the postharvest process, evaluated in the study, provides a considerable contribution to filling the existing information gap, enabling the molecular and phytochemical characterisation of this erosion-endangered fruit. Data show biochemical changes that transform the harvested fruit into palatable consumable product.

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that currently lacks effective therapy. Given the heterogeneity of clinical and molecular profiles of ALS patients, personalized diagnostics and pathological characterization represent a powerful strategy to optimize patient stratification, thereby enabling personalized treatment. Immortalized lymphocytes from sporadic and genetic ALS patients recapitulate some pathological hallmarks of the disease, facilitating the fundamental task of drug screening. However, the molecular aggregation of ALS has not been characterized in this patient-derived cellular model. Indeed, protein aggregation is one of the most prominent features of neurodegenerative diseases, and therefore, models to test drugs against personalized pathological aggregation could help discover improved therapies. With this work, we aimed to characterize the aggregation profile of ALS immortalized lymphocytes and test several drug candidates with different mechanisms of action. In addition, we have evaluated the molecular aggregation in motor neurons derived from two hiPSC cell lines corresponding to ALS patients with different mutations in TARDBP. The results provide valuable insight into the different characterization of sporadic and genetic ALS patients' immortalized lymphocytes, their differential response to drug treatment, and the usefulness of proteome homeostasis characterization in patients' cells.

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With aging, bone mass declines and the anabolic effects of skeletal loading diminish. While much research has focused on gene transcription, how bone ages and loses its mechanoresponsiveness at the protein level remains unclear. We developed a novel proteomics approach and performed a paired mass spectrometry and RNA-seq analysis on tibias from young-adult (5-month) and old (22-month) mice. We report the first correlation estimate between the bone proteome and transcriptome (Spearman ρ = 0.40), which is in line with other tissues but indicates that a relatively low amount of variation in protein levels is explained by the variation in transcript levels. Of 71 shared targets that differed with age, eight were associated with bone mineral density in previous GWAS, including understudied targets Asrgl1 and Timp2. We used complementary RNA in situ hybridization to confirm that Asrgl1 and Timp2 had reduced expression in osteoblasts/osteocytes in old bones. We also found evidence for reduced TGF-beta signaling with aging, in particular Tgfb2. Next, we defined proteomic changes following mechanical loading. At the protein level, bone differed more with age than with loading, and aged bone had fewer loading-induced changes. Overall, our findings underscore the need for complementary protein-level assays in skeletal biology research.

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It was hypothesized that the longissimus thoracis (LT) muscle proteome, phosphoproteome, and metabolome could explain postmortem metabolism and tenderness differences in muscle from cattle supplemented zinc (Zn) and/or ractopamine hydrochloride (RH). High percentage Angus steers (N = 20) were fed in a 2 × 2 factorial assigned to Zn and RH treatments: control (CON; n = 10; analyzed 36 mg Zn/kg dry matter [DM]) or supranutritional Zn supplementation (SUPZN; n = 10; control diet + 60 mg Zn/kg DM [from ZnSO4] + 60 mg Zn/kg DM [from Zn-amino acid complex]) for the entire 89-d trial. During the 28 d before harvest, steers were blocked by body weight within Zn treatments to RH treatments of 0 (NO; n = 10) or 300 mg (RAC; n = 10) per steer per day. Steers were harvested at the Iowa State Meat Laboratory, where pH decline (1, 3, 6, and 24 h postmortem) was measured. At 24 h postmortem, LT muscle sections were removed from carcasses, and steaks were analyzed for Warner-Bratzler shear force (WBSF) values at 1, 3, 7, and 14 d postmortem. Muscle samples were taken at 1 h, 1, 3, 7, and 14 d postmortem for the following analysis: troponin-T degradation (1, 3, 7, and 14 d postmortem), myosin heavy chain analysis (1 h postmortem), sarcoplasmic proteome analysis through tandem mass tagging analysis (1 h and 1 d postmortem), metabolome analysis (1 h and 1 d postmortem), and phosphoproteome analysis (1 h postmortem). SUPZN-NO tended to have a lower (P = 0.06) pH at 6 h postmortem and a lower WBSF value (P = 0.06) at 1 d postmortem. CON-RAC had a higher (P = 0.04) pH at 6 h postmortem and WBSF value (P < 0.01) at 1 d postmortem. A lower pH at 6 h postmortem and lower WBSF value at 1 d postmortem in the SUPZN-NO treatment was accompanied by more sorbitol and fructose at 1 d postmortem, and less myosin regulatory light chain 2 at 1 h postmortem, and less adenosine monophosphate deaminase 1 (AMPD1) at 1 d postmortem than all other treatments. A higher pH at 6 h postmortem and higher WBSF value at 1 d postmortem in CON-RAC and SUPZN-RAC was accompanied by more soluble structural proteins (troponin-T and myosin-7) at 1 h postmortem than CON-NO. At 1 h postmortem, CON-RAC had more glyceraldehyde-3-phosphate dehydrogenase than CON-NO or SUPZN-RAC. Differences in energy metabolism enzymes, metabolites, and structural proteins may affect ATP production, rigor development, and lactate buildup which may explain the differences in postmortem metabolism and tenderness development at 1 d postmortem.

There is significant interest in improving the efficiency of beef production. Beef quality and consumer satisfaction are of equal importance. Feeding zinc (Zn) above nutritional recommendations and feeding ractopamine hydrochloride (RH) can be utilized to optimize cattle growth. However, the impacts of these rapid growth strategies on beef quality are not fully understood. This study aimed to identify variations in the protein and metabolite profile of muscle from cattle-fed Zn and RH that may influence postmortem characteristics of meat. Cattle were fed one of the following diets: control (CON-NO), RH-only supplementation (CON-RAC), supranutritional Zn-only supplementation (SUPZN-NO), and the combination of Zn and RH supplementation (SUPZN-RAC). At harvest, RH supplementation resulted in muscle with a greater pH at 6 h postmortem and increased toughness at 1 d postmortem. Zn supplementation resulted in muscle with a lower pH at 6 h and more tender steaks at 1 d postmortem. Postmortem muscle from cattle supplemented with Zn and RH differed in energy metabolism, stress response, and structural proteins. Further, supplementation led to observed differences in metabolites related to energy production. Variations in the protein and metabolite profiles may have influenced postmortem energy metabolism, impacting pH decline, protein degradation, and tenderness development at 1 d postmortem from cattle supplemented Zn and RH. ZN supplementation may have promise in improving tenderness at 1 d postmortem.

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Leptospires, as motile Gram-negative bacteria, employ sophisticated strategies for efficient invasion and dissemination within their hosts. In response, hosts counteract pathogens through nutritional immunity, a concept involving the deprivation of essential metals such as zinc. Zinc, pivotal in modulating pathogen-host interactions, influences proteins structural, catalytic, and regulatory functions. A comprehensive understanding of how leptospires regulate intracellular zinc availability is crucial for deciphering their survival mechanisms. This study explores the proteomic profile of Leptospira interrogans sv. Copenhageni str. 10A cultivated in Ellinghausen-McCullough-Johnson-Harris medium supplemented with the zinc chelator TPA or ZnCl2. Among the 2161 proteins identified, 488 were subjected to scrutiny, revealing 102 less abundant and 81 more abundant in response to TPA. Of these 488 proteins, 164 were exclusive to the presence of TPA and 141 were exclusive to the zinc-enriched conditions. Differentially expressed proteins were classified into clusters of orthologous groups (COGs) with a distribution in metabolic functions (37.8%), information storage/processing (21.08%), cellular processes/signaling (28.04%), and poorly characterized proteins (10.65%). Differentially expressed proteins are putatively involved in processes like 1-carbon compound metabolism, folate biosynthesis, and amino acid/nucleotide synthesis. Zinc availability significantly impacted key processes putatively related to leptospires' interactions with their host, such as motility, biofilm formation, and immune escape. Under conditions of higher zinc concentration, ribosomal proteins, chaperones and components of transport systems were observed, highlighting interactions between regulatory networks responsive to zinc and iron in L. interrogans. This study not only revealed hypothetical proteins potentially related to zinc homeostasis, but also identified possible virulence mechanisms and pathogen-host adaptation strategies influenced by the availability of this metal. There is an urgent need, based on these data, for further in-depth studies aimed at detailing the role of zinc in these pathways and mechanisms, which may ultimately determine more effective therapeutic approaches to combat Leptospira infections.

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Nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD+), has robust cognitive benefits and alleviates neuroinflammation in Alzheimer's Disease (AD) mouse models without decreasing beta-amyloid plaque pathology. Such effects may be mediated by the reactive species interactome (RSI), at the metabolome level. In this study, we employed in vitro and in vivo models of oxidative stress, aging and AD to profile the effects of NR on neuronal survival, RSI, and the whole proteome characterization of cortex and hippocampus. RSI analysis yielded a complex modulation upon NR treatment. We constructed protein co-expression networks and correlated them to NR treatment and all measured reactive species. We observed brain-area specific effects of NR on co-expressed protein modules of oxidative phosphorylation, fatty acid oxidation, and neurotransmitter regulation pathways, which correlated with RSI components. The current study contributes to the understanding of modulation of the metabolome, specifically after NR treatment in AD and how it may play disease-modifying roles.

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Tendon disorders often result in decreased muscle function and atrophy. Pulsed Electromagnetic Fields (PEMFs) have shown potential in improving tendon fiber structure and muscle recovery. However, the molecular effects of PEMF therapy on skeletal muscle, beyond conventional metrics like MRI or markers of muscle decline, remain largely unexplored. This study investigates the metabolic and structural changes in PEMF-treated muscle tissue using proteomics in a rat model of Achilles tendinopathy induced by collagenase. Sprague Dawley rats were unilaterally induced for tendinopathy with type I collagenase injection and exposed to PEMFs for 8 h/day. Gastrocnemius extracts from untreated or PEMF-treated rats were analyzed with LC-MS/MS, and proteomics differential analysis was conducted through label-free quantitation. PEMF-treated animals exhibited decreased glycolysis and increased LDHB expression, enhancing NAD signaling and ATP production, which boosted respiratory chain activity and fatty acid beta-oxidation. Antioxidant protein levels increased, controlling ROS production. PEMF therapy restored PGC1alpha and YAP levels, decreased by tendinopathy. Additionally, myosins regulating slow-twitch fibers and proteins involved in fiber alignment and force transmission increased, supporting muscle recovery and contractile function. Our findings show that PEMF treatment modulates NAD signaling and oxidative phosphorylation, aiding muscle recovery through the upregulation of YAP and PGC1alpha and increasing slow myosin isoforms, thus speeding up physiological recovery.

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BACKGROUND: Leptin is known for its metabolic, immunomodulatory and neuroendocrine properties, but the full spectrum of molecules downstream of leptin and relevant underlying mechanisms remain to be fully clarified. Our objective was to identify proteins and pathways influenced by leptin through untargeted proteomics in two clinical trials involving leptin administration in lean individuals. METHODS: We performed untargeted liquid chromatography-tandem mass spectrometry serum proteomics across two studies a) Short-term randomized controlled crossover study of lean male and female humans undergoing a 72-h fast with concurrent administration of either placebo or high-dose leptin; b) Long-term (36-week) randomized controlled trial of leptin replacement therapy in human females with acquired relative energy deficiency and hypoleptinemia. We explored longitudinal proteomic changes and run adjusted mixed models followed by post-hoc tests. We further attempted to identify ontological pathways modulated during each experimental condition and/or comparison, through integrated qualitative pathway and enrichment analyses. We also explored dynamic longitudinal relationships between the circulating proteome with clinical and hormonal outcomes. RESULTS: 289 and 357 unique proteins were identified per each respective study. Short-term leptin administration during fasting markedly upregulated several proinflammatory molecules, notably C-reactive protein (CRP) and cluster of differentiation (CD) 14, and downregulated lecithin cholesterol acyltransferase and several immunoglobulin variable chains, in contrast with placebo, which produced minimal changes. Quantitative pathway enrichment further indicated an upregulation of the acute phase response and downregulation of immunoglobulin- and B cell-mediated immunity by leptin. These changes were independent of participants' biological sex. In the long term study, leptin likewise robustly and persistently upregulated proteins of the acute phase response, and downregulated immunoglobulin-mediated immunity. Leptin also significantly and differentially affected a wide array of proteins related to immune function, defense response, coagulation, and inflammation compared with placebo. These changes were more notable at the 24-week visit, coinciding with the highest measured levels of serum leptin. We further identified distinct co-regulated clusters of proteins and clinical features during leptin administration indicating robust longitudinal correlations between the regulation of immunoglobulins, immune-related molecules, serpins (including cortisol and thyroxine-binding globulins), lipid transport molecules and growth factors, in contrast with placebo, which did not produce similar associations. CONCLUSIONS: These high-throughput longitudinal results provide unique functional insights into leptin physiology, and pave the way for affinity-based proteomic analyses measuring several thousands of molecules, that will confirm these data and may fully delineate underlying mechanisms.

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The interactions of different dietary doses of copper with fructose contribute to the development of metabolic dysfunction-associated steatotic liver disease (MASLD) via the gut-liver axis. The underlying mechanisms remain elusive. The aim of this study was to identify the specific pathways leading to gut barrier dysfunction in the ileum using a proteomics approach in a rat model. Male weanling Sprague Dawley rats were fed diets with adequate copper (CuA), marginal copper (CuM), or supplemented copper (CuS) in the absence or presence of fructose supplementation (CuAF, CuMF, and CuSF) for 4 weeks. Ileum protein was extracted and analyzed with an LC-MS. A total of 2847 differentially expressed proteins (DEPs) were identified and submitted to functional enrichment analysis. As a result, the ileum proteome and signaling pathways that were differentially altered were revealed. Of note, the CuAF is characterized by the enrichment of oxidative phosphorylation and ribosome as analyzed with the KEGG; the CuMF is characterized by an enriched arachidonic acid metabolism pathway; and focal adhesion, the regulation of the actin cytoskeleton, and tight junction were significantly enriched by the CuSF. In conclusion, our proteomics analysis identified the specific pathways in the ileum related to the different dietary doses of copper-fructose interactions, suggesting that distinct mechanisms in the gut are involved in the development of MASLD.