CCDC78: Unveiling the Function of a Novel Gene Associated with Hereditary Myopathy.

CCDC78 was identified as a novel candidate gene for autosomal dominant centronuclear myopathy-4 (CNM4) approximately ten years ago. However, to date, only one family has been described, and the function of CCDC78 remains unclear. Here, we analyze for the first time a family harboring a CCDC78 nonsense mutation to better understand the role of CCDC78 in muscle. METHODS: We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined CCDC78 transcripts and protein using WB in CCDC78-mutated muscle tissue; these analyses were also performed on muscle, lymphocytes, and fibroblasts from healthy subjects. Subsequently, we conducted RT-qPCR and transcriptome profiling through RNA-seq to evaluate changes in gene expression associated with CCDC78 dysfunction in muscle. Lastly, coimmunoprecipitation (Co-Ip) assays and mass spectrometry (LC-MS/MS) studies were carried out on extracted muscle proteins from both healthy and mutated subjects. RESULTS: The histopathological features in muscle showed novel histological hallmarks, which included areas of dilated and swollen sarcoplasmic reticulum (SR). We provided evidence of nonsense-mediated mRNA decay (NMD), identified the presence of novel CCDC78 transcripts in muscle and lymphocytes, and identified 1035 muscular differentially expressed genes, including several involved in the SR. Through the Co-Ip assays and LC-MS/MS studies, we demonstrated that CCDC78 interacts with two key SR proteins: SERCA1 and CASQ1. We also observed interactions with MYH1, ACTN2, and ACTA1. CONCLUSIONS: Our findings provide insight, for the first time, into the interactors and possible role of CCDC78 in skeletal muscle, locating the protein in the SR. Furthermore, our data expand on the phenotype previously associated with CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4.

Subchronic Effects of Tetrachloroethylene on Two Freshwater Copepod Species: Implications for Groundwater Risk Assessment.

Aliphatic chlorinated hydrocarbons, notably tetrachloroethylene (also known as perchloroethylene [PCE]), are persistent, mobile, and toxic (PMT) and/or very persistent, mobile, and toxic (vPMT) groundwater pollutants, often exceeding safe drinking water thresholds. The present study delves into the groundwater risk assessment of PCE with a novel focus on the sensitivity of stygobitic species-organisms uniquely adapted to groundwater environments. Through a comparative analysis of the subchronic effects of PCE on the locomotion behavior of two copepod species, the stygobitic Moraria sp. and the nonstygobitic Bryocamptus zschokkei, we highlighted the inadequacy of the current European predicted-no-effect concentration of PCE for groundwater ecosystems. Our findings indicate significant behavioral impairments in both species at a concentration (32 ng/L PCE) well below the threshold deemed safe, suggesting that the current European guidelines for groundwater risk assessment may not adequately protect the unique biodiversity of groundwater habitats. Importantly, B. zschokkei demonstrated sensitivity to PCE comparable to or greater than that of the target stygobitic species, suggesting its utility as a substitute species in groundwater risk assessment. The present study adds to the limited research on the ecotoxicological sensitivity of groundwater species to PMT/vPMT chemicals and highlights the need for refined groundwater risk-assessment methodologies that consider the susceptibilities of stygobitic species. Environ Toxicol Chem 2024;00:1-13. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The immune and microbial homeostasis determines the Candida-mast cells cross-talk in celiac disease.

Celiac disease (CD) is an autoimmune enteropathy resulting from an interaction between diet, genome, and immunity. Although many patients respond to a gluten-free diet, in a substantive number of individuals, the intestinal injury persists. Thus, other factors might amplify the ongoing inflammation. Candida albicans is a commensal fungus that is well adapted to the intestinal life. However, specific conditions increase Candida pathogenicity. The hypothesis that Candida may be a trigger in CD has been proposed after the observation of similarity between a fungal wall component and two CD-related gliadin T-cell epitopes. However, despite being implicated in intestinal disorders, Candida may also protect against immune pathologies highlighting a more intriguing role in the gut. Herein, we postulated that a state of chronic inflammation associated with microbial dysbiosis and leaky gut are favorable conditions that promote C. albicans pathogenicity eventually contributing to CD pathology via a mast cells (MC)-IL-9 axis. However, the restoration of immune and microbial homeostasis promotes a beneficial C. albicans-MC cross-talk favoring the attenuation of CD pathology to alleviate CD pathology and symptoms.

A microbially produced AhR ligand promotes a Tph1-driven tolerogenic program in multiple sclerosis.

Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases.

SARS-CoV-2 Infection is Associated with Age- and Gender-Specific Changes in the Nasopharyngeal Microbiome.

BACKGROUND: The recent Coronavirus Disease 2019 (COVID-19) pandemic has dramatically exposed our gap in understanding the pathogenesis of airborne infections. Within such a context, it is increasingly clear that the nasal cavity represents a critical checkpoint not only in the initial colonization phase but also in shaping any infectious sequelae. This is particularly relevant to COVID-19 in that the nasal cavity is characterized by high-level expression of the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) receptor, Angiotensin-Converting Enzyme 2 (ACE2), all along the respiratory tract. As part of the nasal mucosa, commensal microbes harbored by the nasal cavity likely are far more than just innocent bystanders in the interaction between SARS-CoV-2 and the local microenvironment. Yet the role of the qualitative composition of the nasal microbiome is unclear, as is its function, whether protective or not. METHODS: In this study, individuals undergoing SARS-CoV-2 molecular testing at the Hospital of Perugia (Italy) were recruited, with their residual material from the nasopharyngeal swabs being collected for microbiome composition analysis and short-chain fatty acid (SCFA) measurements (by 16S rRNA sequencing and gas chromatography-mass spectrometry), respectively. RESULTS: After stratification by age, gender, and viral load, the composition of the nasopharyngeal microbiome appeared to be influenced by age and gender, and SARS-CoV-2 infection further determined compositional changes. Notwithstanding this variability, a restricted analysis of female subjects-once SARS-CoV-2-infected-unraveled a shared expansion of Lachnospirales-Lachnospiraceae, irrespective of the viral load and age. This was associated with a reduction in the branched SCFA isobutanoic acid, as well as in the SCFAs with longer chains. CONCLUSIONS: Our results indicate that the nasopharyngeal microbiome is influenced by age, gender, and viral load, with consistent patterns of microbiome changes being present across specific groups. This may help in designing a personalized medicine approach in COVID-19 patients with specific patterns of nasal microbial communities.

CPX-351 exploits the gut microbiota to promote mucosal barrier function, colonization resistance, and immune homeostasis.

ABSTRACT: CPX-351, a liposomal combination of cytarabine plus daunorubicin, has been approved for the treatment of adults with newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes, because it improves survival and outcome of patients who received hematopoietic stem cell transplant compared with the continuous infusion of cytarabine plus daunorubicin (referred to as "7 + 3" combination). Because gut dysbiosis occurring in patients with AML during induction chemotherapy heavily affects the subsequent phases of therapy, we have assessed whether the superior activity of CPX-351 vs "7 + 3" combination in the real-life setting implicates an action on and by the intestinal microbiota. To this purpose, we have evaluated the impact of CPX-351 and "7 + 3" combination on mucosal barrier function, gut microbial composition and function, and antifungal colonization resistance in preclinical models of intestinal damage in vitro and in vivo and fecal microbiota transplantation. We found that CPX-351, at variance with "7 + 3" combination, protected from gut dysbiosis, mucosal damage, and gut morbidity while increasing antifungal resistance. Mechanistically, the protective effect of CPX-351 occurred through pathways involving both the host and the intestinal microbiota, namely via the activation of the aryl hydrocarbon receptor-interleukin-22 (IL-22)-IL-10 host pathway and the production of immunomodulatory metabolites by anaerobes. This study reveals how the gut microbiota may contribute to the good safety profile, with a low infection-related mortality, of CPX-351 and highlights how a better understanding of the host-microbiota dialogue may contribute to pave the way for precision medicine in AML.

Clinical manifestations and analytical reports for MDPHP acute intoxication cases.

MDPHP is a synthetic cathinone (SC) belonging to α-pyrrolidinophenone derivatives. It is a central nervous system stimulant and may induce hallucinations, paranoia, tachycardia, hypertension, chest pain, and rhabdomyolysis. In literature, a few cases of intoxication have been reported. In the present study, 17 cases of MDPHP intake were described including the analytical findings and clinical manifestations. MDPHP was quantified by liquid chromatography-tandem mass spectrometry in blood (range 1.26-73.30 ng/mL) and urine (range 19.31-8769.64 ng/mL) samples. In three cases the presence of α-PHP was observed. In one case, MDPHP was the only detected substance. Concomitant use of MDPHP with other substances, particularly psychostimulants, was common and it was difficult to describe the peculiar clinical characteristics of this SC. Most of the symptoms overlapped those expected, some of them were unusual and all of them particularly severe thus inducing the research of NPS in laboratory tests. We demonstrated the presence of psychiatric, neurological, and respiratory symptoms, as well as the possible presence of rhabdomyolysis and cardiotoxicity associated with the use of MDPHP. ED admissions were also more frequent in patients with addiction problems. In some cases, MDPHP intake required intensive supportive care. A multidisciplinary approach, including specialist consultation, is recommended for patients showing challenging features. Moreover, we demonstrated that the adoption of advanced analytical techniques, i.e., liquid chromatography-tandem mass spectrometry, is necessary to detect these molecules. Further studies are needed to understand MDPHP intake patterns and associated symptoms. It is essential to raise awareness in addiction treatment centers and among potential users, especially young people, and chemsex addicted.

Quantitative Colorimetric Sensing of Carbidopa in Anti-Parkinson Drugs Based on Selective Reaction with Indole-3-Carbaldehyde.

The quality of life of patients affected by Parkinson's disease is improved by medications containing levodopa and carbidopa, restoring the dopamine concentration in the brain. Accordingly, the affordable quality control of such pharmaceuticals is very important. Here is reported the simple and inexpensive colorimetric quantification of carbidopa in anti-Parkinson drugs by the selective condensation reaction between the hydrazine group from carbidopa and the formyl functional group of selected aldehydes in acidified hydroalcoholic solution. An optical assay was developed by using indole-3-carbaldehyde (I3A) giving a yellow aldazine in EtOH:H2O 1:1 (λmax~415 nm) at 70 °C for 4 h, as confirmed by LC-MS analysis. A filter-based plate reader was used for colorimetric data acquisition, providing superior results in terms of analytical performances for I3A, with a sensitivity ~50 L g-1 and LOD ~0.1 mg L-1 in comparison to a previous study based on vanillin, giving, for the same figures of merit values, about 13 L g-1 and 0.2-0.3 mg L-1, respectively. The calibration curves for the standard solution and drugs were almost superimposable, therefore excluding interference from the excipients and additives, with very good reproducibility (avRSD% 2-4%) within the linear dynamic range (10 mg L-1-50 mg L-1).

Using wasps as a tool to restore a functioning vine grape mycobiota and preserve the mycobial "terroir".

In the last one-hundred years, the exponential expansion of wine making has artificialized the agricultural landscape as well as its microbial diversity, spreading human selected Saccharomyces cerevisiae strains. Evidence showed that social wasps can harbor a significant fraction of the yeast phenotypic diversity of a given area of wine production, allowing different strains to overwinter and mate in their gut. The integrity of the wasp-yeast ecological interaction is of paramount importance to maintain the resilience of microbial populations associated to wine aromatic profiles. In a field experiment, we verified whether Polistes dominula wasps, reared in laboratory and fed with a traceable S. cerevisiae strain, could be a useful tool to drive the controlled yeast dispersion directly on grapes. The demonstration of the biotechnological potential of social insects in organic wine farming lays the foundations for multiple applications including maintenance of microbial biodiversity and rewilding vineyards through the introduction of wasp associated microbiomes.

Inflammation, mitochondrial dysfunction and physical performance: a possible association in older patients with persistent atrial fibrillation-the results of a preliminary study.

BACKGROUND: Atrial fibrillation (AF) is associated with chronic inflammation, a hallmark of ageing process. The aim of this study was to determine interleukin-6 (IL-6)-associated variables, also exploring acylcarnitines, expression of mitochondrial abnormalities. METHODS: We evaluated 22 controls and 50 patients with persistent AF. IL-6 and acylcarnitines were measured with ELISA kits and mass spectrometry techniques. RESULTS: IL-6 concentration (mean: 3.9 ± 3.1 pg/mL) was lower in controls and increased in AF patients, especially with heart failure. The CHA2DS2-VASc, the MMSE and the SPPB scores were 3.8 ± 1.6, 28 ± 2 and 9.4 ± 2.1. Thirteen acylcanitines correlated with IL-6. At multivariable analysis, IL-6 was directly associated with C4-OH-a short-chain acylcarnitine, fibrinogen and alanine aminotransferase values, and with hyperuricemia. An inverse association existed with calcium concentration and SPPB score. CONCLUSIONS: In older AF patients, IL-6 correlated with acylcarnitines and lower physical performance. Alterations in energy production, reduced physical function and inflammation could contribute to frailty development.

Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays.

BACKGROUND: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. METHODS: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. RESULTS: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. CONCLUSIONS: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.

Salt-Affected Rocket Plants as a Possible Source of Glucosinolates.

Soil salinity can have various negative consequences on agricultural products, from their quality and production to their aesthetic traits. In this work, the possibility to use salt-affected vegetables, that otherwise would be discarded, as a source of nutraceuticals was explored. To this aim, rocket plants, a vegetable featuring bioactive compounds such as glucosinolates, were exposed to increasing NaCl concentrations in hydroponics and analysed for their content in bioactive compounds. Salt levels higher than 68 mM produced rocket plants that did not comply with European Union regulations and would therefore be considered a waste product. Anyway, our findings, obtained by Liquid Chromatography-High Resolution Mass Spectrometry, demonstrated a significant increase in glucosinolates levels in such salt-affected plants. opening the opportunity for a second life of these market discarded products to be recycled as glucosinolates source. Furthermore, an optimal situation was found at NaCl 34 mM in which not only were the aesthetic traits of rocket plants not affected, but also the plants revealed a significant enrichment in glucosinolates. This can be considered an advantageous situation in which the resulting vegetables still appealed to the market and showed improved nutraceutical aspects.

Millet Fermented by Different Combinations of Yeasts and Lactobacilli: Effects on Phenolic Composition, Starch, Mineral Content and Prebiotic Activity.

Millet is the sixth-highest yielding grain in the world and a staple crop for millions of people. Fermentation was applied in this study to improve the nutritional properties of pearl millet. Three microorganism combinations were tested: Saccharomyces boulardii (FPM1), Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius (FPM2) and Hanseniaspora uvarum plus Fructilactobacillus sanfranciscensis (FPM3). All the fermentation processes led to an increase in minerals. An increase was observed for calcium: 254 ppm in FPM1, 282 ppm in FPM2 and 156 ppm in the unfermented sample. Iron increased in FPM2 and FPM3 (approx. 100 ppm) with respect the unfermented sample (71 ppm). FPM2 and FPM3 resulted in richer total phenols (up to 2.74 mg/g) compared to the unfermented sample (2.24 mg/g). Depending on the microorganisms, it was possible to obtain different oligopeptides with a mass cut off ≤10 kDalton that was not detected in the unfermented sample. FPM2 showed the highest resistant starch content (9.83 g/100 g) and a prebiotic activity on Bifidobacterium breve B632, showing a significant growth at 48 h and 72 h compared to glucose (p < 0.05). Millet fermented with Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius can be proposed as a new food with improved nutritional properties to increase the quality of the diet of people who already use millet as a staple food.

Venom Collection by Electrical Stimulation in the Invasive Species Polistes dominula Reared Using a Vespiculture Regime.

Specific Venom Immunotherapy (VIT) is practiced with venom extracted from insects, and is the specific therapy used for patients highly allergic to social insect (Hymenoptera) stings. Due to the dramatic shortage of vespid species in the local environment, we coupled vespiculture techniques of Polistes paper wasps with a venom collection procedure based on the electrical stimulation of individuals from entire colonies. The procedure involves little to no disturbance of the individual insects, and at the same time, successfully allows for the extraction of venom containing all allergens necessary for VIT.

Studying the trafficking of labeled trodusquemine and its application as nerve marker for light-sheet and expansion microscopy.

Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.

Steryl ferulates composition in twenty-two millet samples: Do "microwave popping" and fermentation affect their content?

Climate change has led to rediscovery of minor drought tolerant grains such as millet. Among its bioactive molecules, steryl ferulates have been poorly explored. Steryl ferulates composition of was investigated by high performance liquid chromatography-diode array detector-tandem mass spectrometry and high resolution tandem mass spectrometry in twenty-two millet samples and also in some fermented and microwave heated samples. Six compounds were found in Panicum, Pennisetum, Eleusine and Setaria genera, with a prevalence of campestanyl and sitostanyl ferulate. The lowest steryl ferulates content was found in Panicum, with values ranging from 2.98 ± 0.04 µg/g to 8.72 ± 0.41 µg/g. Foxtail millet and finger millet showed the highest amount with 46.07 ± 5.20 µg/g and 85.29 ± 4.30 µg/g, respectively. As for pearl millet, microwave heating and fermentation increased steryl ferulates by two (33.77 ± 0.88 µg/g) and five (75.83 ± 1.25 µg/g) times, with respect to the untreated sample. Microwave heating and fermentation could be used to increase steryl ferulates in millet.

Optimizing therapeutic outcomes of immune checkpoint blockade by a microbial tryptophan metabolite.

BACKGROUND: Despite the great success, the therapeutic benefits of immune checkpoint inhibitors (ICIs) in cancer immunotherapy are limited by either various resistance mechanisms or ICI-associated toxic effects including gastrointestinal toxicity. Thus, novel therapeutic strategies that provide manageable side effects to existing ICIs would enhance and expand their therapeutic efficacy and application. Due to its proven role in cancer development and immune regulation, gut microbiome has gained increasing expectation as a potential armamentarium to optimize immunotherapy with ICI. However, much has to be learned to fully harness gut microbiome for clinical applicability. Here we have assessed whether microbial metabolites working at the interface between microbes and the host immune system may optimize ICI therapy. METHODS: To this purpose, we have tested indole-3-carboxaldehyde (3-IAld), a microbial tryptophan catabolite known to contribute to epithelial barrier function and immune homeostasis in the gut via the aryl hydrocarbon receptor (AhR), in different murine models of ICI-induced colitis. Epithelial barrier integrity, inflammation and changes in gut microbiome composition and function were analyzed. AhR, indoleamine 2,3-dioxygenase 1, interleukin (IL)-10 and IL-22 knockout mice were used to investigate the mechanism of 3-IAld activity. The function of the microbiome changes induced by 3-IAld was evaluated on fecal microbiome transplantation (FMT). Finally, murine tumor models were used to assess the effect of 3-IAld treatment on the antitumor activity of ICI. RESULTS: On administration to mice with ICI-induced colitis, 3-IAld protected mice from intestinal damage via a dual action on both the host and the microbes. Indeed, paralleling the activation of the host AhR/IL-22-dependent pathway, 3-IAld also affected the composition and function of the microbiota such that FMT from 3-IAld-treated mice protected against ICI-induced colitis with the contribution of butyrate-producing bacteria. Importantly, while preventing intestinal damage, 3-IAld did not impair the antitumor activity of ICI. CONCLUSIONS: This study provides a proof-of-concept demonstration that moving past bacterial phylogeny and focusing on bacterial metabolome may lead to a new class of discrete molecules, and that working at the interface between microbes and the host immune system may optimize ICI therapy.

A Shifted Composition of the Lung Microbiota Conditions the Antifungal Response of Immunodeficient Mice.

The microbiome, i.e., the communities of microbes that inhabit the surfaces exposed to the external environment, participates in the regulation of host physiology, including the immune response against pathogens. At the same time, the immune response shapes the microbiome to regulate its composition and function. How the crosstalk between the immune system and the microbiome regulates the response to fungal infection has remained relatively unexplored. We have previously shown that strict anaerobes protect from infection with the opportunistic fungus Aspergillus fumigatus by counteracting the expansion of pathogenic Proteobacteria. By resorting to immunodeficient mouse strains, we found that the lung microbiota could compensate for the lack of B and T lymphocytes in Rag1-/- mice by skewing the composition towards an increased abundance of protective anaerobes such as Clostridia and Bacteroidota. Conversely, NSG mice, with major defects in both the innate and adaptive immune response, showed an increased susceptibility to infection associated with a low abundance of strict anaerobes and the expansion of Proteobacteria. Further exploration in a murine model of chronic granulomatous disease, a primary form of immunodeficiency characterized by defective phagocyte NADPH oxidase, confirms the association of lung unbalance between anaerobes and Proteobacteria and the susceptibility to aspergillosis. Consistent changes in the lung levels of short-chain fatty acids between the different strains support the conclusion that the immune system and the microbiota are functionally intertwined during Aspergillus infection and determine the outcome of the infection.

Surfactant-Assisted Distal Pulmonary Distribution of Budesonide Revealed by Mass Spectrometry Imaging.

Direct lung administration of budesonide in combination with surfactant reduces the incidence of bronchopulmonary dysplasia. Although the therapy is currently undergoing clinical development, the lung distribution of budesonide throughout the premature neonatal lung has not yet been investigated. Here, we applied mass spectrometry imaging (MSI) to investigate the surfactant-assisted distal lung distribution of budesonide. Unlabeled budesonide was either delivered using saline as a vehicle (n = 5) or in combination with a standard dose of the porcine surfactant Poractant alfa (n = 5). These lambs were ventilated for one minute, and then the lungs were extracted for MSI analysis. Another group of lambs (n = 5) received the combination of budesonide and Poractant alfa, followed by two hours of mechanical ventilation. MSI enabled the label-free detection and visualization of both budesonide and the essential constituent of Poractant alfa, the porcine surfactant protein C (SP-C). 2D ion intensity images revealed a non-uniform distribution of budesonide with saline, which appeared clustered in clumps. In contrast, the combination therapy showed a more homogeneous distribution of budesonide throughout the sample, with more budesonide distributed towards the lung periphery. We found similar distribution patterns for the SP-C and budesonide in consecutive lung tissue sections, indicating that budesonide was transported across the lungs associated with the exogenous surfactant. After two hours of mechanical ventilation, the budesonide intensity signal in the 2D ion intensity maps dropped dramatically, suggesting a rapid lung clearance and highlighting the relevance of achieving a uniform surfactant-assisted lung distribution of budesonide early after delivery to maximize the anti-inflammatory and maturational effects throughout the lung.

Sample preparation strategy for the detection of steroid-like compounds using MALDI mass spectrometry imaging: pulmonary distribution of budesonide as a case study.

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard's reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples.