Exploring the Effects of Cyclosporin A to Isocyclosporin A Rearrangement on Ion Mobility Separation.

Cyclosporin A (CycA) is a peptide secondary metabolite derived from fungi that plays a crucial role in transplantation surgery. Cyclic traveling wave ion mobility mass spectrometry (IM-MS) revealed an N → O peptidyl shift in singly protonated CycA to isocyclosporin A (isoA), whereas no such isomerization was observed for doubly protonated and sodiated molecules. CycA and isoA were able to be separated by considering doubly protonated precursors using a specific ion fragment. In parallel, sodium ion stabilization facilitated the simultaneous separation and quantitation of singly charged cyclosporin isomers with the limit of detection and coefficient of determination of 1.3% and 0.9908 for CycA in isoA and 1.0% and 0.9830 for isoA in CycA, respectively. Finally, 1H-13C gHSQC NMR experiments permitted parallel recording of up to 11 cyclosporin conformers. The ratios were determined by integrating the volume of cross-peaks of the upfield resonating hydrogen in the diastereotopic methylene group of sarcosine-3.

The Deciphering of Growth-Dependent Strategies for Quorum-Sensing Networks in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is recognized as a significant cause of morbidity and mortality among nosocomial pathogens. In respiratory infections, P. aeruginosa acts not only as a single player but also collaborates with the opportunistic fungal pathogen Aspergillus fumigatus. This study introduced a QS molecule portfolio as a potential new biomarker that affects the secretion of virulence factors and biofilm formation. The quantitative levels of QS molecules, including 3-o-C12-HSL, 3-o-C8-HSL, C4-HSL, C6-HSL, HHQ, PQS, and PYO, measured using mass spectrometry in a monoculture, indicated metabolic changes during the transition from planktonic to sessile cells. In the co-cultures with A. fumigatus, the profile of abundant QS molecules was reduced to 3-o-C12-HSL, C4-HSL, PQS, and PYO. A decrease in C4-HSL by 50% to 170.6 ± 11.8 ng/mL and an increase 3-o-C12-HSL by 30% up to 784.4 ± 0.6 ng/mL were detected at the stage of the coverage of the hyphae with bacteria. Using scanning electron microscopy, we showed the morphological stages of the P. aeruginosa biofilm, such as cell aggregates, maturated biofilm, and cell dispersion. qPCR quantification of the genome equivalents of both microorganisms suggested that they exhibited an interplay strategy rather than antagonism. This is the first study demonstrating the quantitative growth-dependent appearance of QS molecule secretion in a monoculture of P. aeruginosa and a co-culture with A. fumigatus.

Spatial lipidomics reveals brain region-specific changes of sulfatides in an experimental MPTP Parkinson's disease primate model.

Metabolism of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to the neurotoxin MPP+ in the brain causes permanent Parkinson's disease-like symptoms by destroying dopaminergic neurons in the pars compacta of the substantia nigra in humans and non-human primates. However, the complete molecular pathology underlying MPTP-induced parkinsonism remains poorly understood. We used dual polarity matrix-assisted laser desorption/ionization mass spectrometry imaging to thoroughly image numerous glycerophospholipids and sphingolipids in coronal brain tissue sections of MPTP-lesioned and control non-human primate brains (Macaca mulatta). The results revealed specific distributions of several sulfatide lipid molecules based on chain-length, number of double bonds, and importantly, hydroxylation stage. More specifically, certain long-chain hydroxylated sulfatides with polyunsaturated chains in the molecular structure were depleted within motor-related brain regions in the MPTP-lesioned animals, e.g., external and internal segments of globus pallidus and substantia nigra pars reticulata. In contrast, certain long-chain non-hydroxylated sulfatides were found to be elevated within the same brain regions. These findings demonstrate region-specific dysregulation of sulfatide metabolism within the MPTP-lesioned macaque brain. The depletion of long-chain hydroxylated sulfatides in the MPTP-induced pathology indicates oxidative stress and oligodendrocyte/myelin damage within the pathologically relevant brain regions. Hence, the presented findings improve our current understanding of the molecular pathology of MPTP-induced parkinsonism within primate brains, and provide a basis for further research regarding the role of dysregulated sulfatide metabolism in PD.

[68Ga]Ga-Ornibactin for Burkholderia cepacia complex Infection Imaging Using Positron Emission Tomography.

Bacteria from the Burkholderia cepacia complex are generally considered to be non-pathogenic to the healthy population. However, some of these species may cause serious nosocomial infections in immunocompromised patients; as such, it is essential to diagnose these infections rapidly so that adequate treatment can be initiated. We report here the use of a radiolabeled siderophore, ornibactin (ORNB), for positron emission tomography imaging. We successfully radiolabeled ORNB with gallium-68 with high radiochemical purity and proved that the resulting complex has optimal in vitro characteristics. In mice, the complex did not show excessive accumulation in organs and was excreted in the urine. We demonstrated that the [68Ga]Ga-ORNB complex accumulates at the site of Burkholderia multivorans infection, including pneumonia, in two animal infection models. These results suggest that [68Ga]Ga-ORNB is a promising tool for the diagnosis, monitoring, and evaluation of the therapeutic response to B. cepacia complex infection.

Siderophore-Based Noninvasive Differentiation of Aspergillus fumigatus Colonization and Invasion in Pulmonary Aspergillosis.

Germination from conidia to hyphae and hyphal propagation of Aspergillus fumigatus are the key pathogenic steps in the development of invasive pulmonary aspergillosis (IPA). By applying in vitro observations in a clinical study of 13 patients diagnosed with probable IPA, here, we show that the transition from colonization to the A. fumigatus invasive stage is accompanied by the secretion of triacetylfusarinine C (TafC), triacetylfusarinine B (TafB), and ferricrocin (Fc) siderophores into urine, with strikingly better sensitivity performance than serum sampling. The best-performing index, the TafC/creatinine index, with a median value of 17.2, provided 92.3% detection sensitivity (95% confidence interval [CI], 64.0 to 99.8%) and 100% specificity (95% CI, 84.6 to 100%), i.e., substantially better than the corresponding indications provided by galactomannan (GM) and ß-d-glucan (BDG) serology. For the same patient cohort, the serum GM and BDG sensitivities were 46.2 and 76.9%, respectively, and their specificities were 86.4 and 63.6%, respectively. The time-dependent specific appearance of siderophores in the host's urine represents an impactful clinical diagnostic advantage in the early discrimination of invasive aspergillosis from colonization. A favorable concentration of TafC in a clinical specimen distant from a deep infection site enables the noninvasive sampling of patients suffering from IPA. IMPORTANCE The importance of this research lies in the demonstration that siderophore analysis can distinguish between asymptomatic colonization and invasive pulmonary aspergillosis. We found clear associations between phases of fungal development, from conidial germination to the proliferative stage of invasive aspergillosis, and changes in secondary metabolite secretion. The critical extracellular fungal metabolites triacetylfusarinines C and B are produced during the polarized germination or postpolarized growth phase and reflect the morphological status of the proliferating pathogen. False positivity in Aspergillus diagnostics is minimized as mammalian cells do not synthesize Aspergillus siderophore or mycotoxin molecules.

Infection metallomics for critical care in the post-COVID era.

Infection metallomics is a mass spectrometry (MS) platform we established based on the central concept that microbial metallophores are specific, sensitive, noninvasive, and promising biomarkers of invasive infectious diseases. Here we review the in vitro, in vivo, and clinical applications of metallophores from historical and functional perspectives, and identify under-studied and emerging application areas with high diagnostic potential for the post-COVID era. MS with isotope data filtering is fundamental to infection metallomics; it has been used to study the interplay between "frenemies" in hosts and to monitor the dynamic response of the microbiome to antibiotic and antimycotic therapies. During infection in critically ill patients, the hostile environment of the host's body activates secondary bacterial, mycobacterial, and fungal metabolism, leading to the production of metallophores that increase the pathogen's chance of survival in the host. MS can reveal the structures, stability, and threshold concentrations of these metal-containing microbial biomarkers of infection in humans and model organisms, and can discriminate invasive disease from benign colonization based on well-defined thresholds distinguishing proliferation from the colonization steady state.

Neuropharmacokinetic visualization of regional and subregional unbound antipsychotic drug transport across the blood-brain barrier.

Comprehensive determination of the extent of drug transport across the region-specific blood-brain barrier (BBB) is a major challenge in preclinical studies. Multiple approaches are needed to determine the regional free (unbound) drug concentration at which a drug engages with its therapeutic target. We present an approach that merges in vivo and in vitro neuropharmacokinetic investigations with mass spectrometry imaging to quantify and visualize both the extent of unbound drug BBB transport and the post-BBB cerebral distribution of drugs at regional and subregional levels. Direct imaging of the antipsychotic drugs risperidone, clozapine, and olanzapine using this approach enabled differentiation of regional and subregional BBB transport characteristics at 20-µm resolution in small brain regions, which could not be achieved by other means. Our approach allows investigation of heterogeneity in BBB transport and presents new possibilities for molecular psychiatrists by facilitating interpretation of regional target-site exposure results and decision-making.

Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs.

A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.

Synthesis of the Hydroxamate Siderophore Nα-Methylcoprogen B in Scedosporium apiospermum Is Mediated by sidD Ortholog and Is Required for Virulence.

Scedosporium species rank second among the filamentous fungi capable to colonize chronically the respiratory tract of patients with cystic fibrosis (CF). Nevertheless, there is little information on the mechanisms underpinning their virulence. Iron acquisition is critical for the growth and pathogenesis of many bacterial and fungal genera that chronically inhabit the CF lungs. In a previous study, we showed the presence in the genome of Scedosporium apiospermum of several genes relevant for iron uptake, notably SAPIO_CDS2806, an ortholog of sidD, which drives the synthesis of the extracellular hydroxamate-type siderophore fusarinine C (FsC) and its derivative triacetylfusarinine C (TAFC) in Aspergillus fumigatus. Here, we demonstrate that Scedosporium apiospermum sidD gene is required for production of an excreted siderophore, namely, Nα-methylcoprogen B, which also belongs to the hydroxamate family. Blockage of the synthesis of Nα-methylcoprogen B by disruption of the sidD gene resulted in the lack of fungal growth under iron limiting conditions. Still, growth of ΔsidD mutants could be restored by supplementation of the culture medium with a culture filtrate from the parent strain, but not from the mutants. Furthermore, the use of xenosiderophores as the sole source of iron revealed that S. apiospermum can acquire the iron using the hydroxamate siderophores ferrichrome or ferrioxamine, i.e., independently of Nα-methylcoprogen B production. Conversely, Nα-methylcoprogen B is mandatory for iron acquisition from pyoverdine, a mixed catecholate-hydroxamate siderophore. Finally, the deletion of sidD resulted in the loss of virulence in a murine model of scedosporiosis. Our findings demonstrate that S. apiospermum sidD gene drives the synthesis of a unique extracellular, hydroxamate-type iron chelator, which is essential for fungal growth and virulence. This compound scavenges iron from pyoverdine, which might explain why S. apiospermum and Pseudomonas aeruginosa are rarely found simultaneously in the CF lungs.

Analysis of Microbial Siderophores by Mass Spectrometry.

Siderophores represent important microbial virulence factors and infection biomarkers. Their monitoring in fermentation broths, bodily fluids, and tissues should be reproducible. Similar isolation, characterization, and quantitation studies can often have conflicting results, and without proper documentation of sample collection, data processing, and analysis methods, it is difficult to reexamine the data and reconcile these differences. In this Springer Nature Protocol, we present the procedure optimized for ferricrocin/triacetylfusarinine C extraction from biological material as well as for tissue fixation and cryosectioning for optical microscopy and for both elemental and molecular mass spectrometry imaging. Special attention is paid to siderophore data mining from conventional and product ion mass spectra, liquid chromatography, and mass spectrometry imaging datasets, performed here by our free software called CycloBranch.

Membrane depolarization and aberrant lipid distributions in the neonatal rat brain following hypoxic-ischaemic insult.

Neonatal hypoxic-ischaemic (HI) encephalopathy is among the most serious complications in neonatology. In the present study, we studied the immediate (0 hour), subacute (36 hours) and late (144 hours) responses of the neonatal brain to experimental HI insult in laboratory rats. At the striatal level, the mass spectrometry imaging revealed an aberrant plasma membrane distribution of Na+/K+ ions in the oedema-affected areas. The failure of the Na+/K+ gradients was also apparent in the magnetic resonance imaging measurements, demonstrating intracellular water accumulation during the acute phase of the HI insult. During the subacute phase, compared with the control brains, an incipient accumulation of an array of N-acylphosphatidylethanolamine (NAPE) molecules was detected in the HI-affected brains, and both the cytotoxic and vasogenic types of oedema were detected. In the severely affected brain areas, abnormal distributions of the monosialogangliosides GM2 and GM3 were observed in two-thirds of the animals exposed to the insult. During the late stage, a partial restoration of the brain tissue was observed in most rats in both the in vivo and ex vivo studies. These specific molecular changes may be further utilized in neonatology practice in proposing and testing novel therapeutic strategies for the treatment of neonatal HI encephalopathy.

Non-invasive and invasive diagnoses of aspergillosis in a rat model by mass spectrometry.

Invasive pulmonary aspergillosis results in 450,000 deaths per year and complicates cancer chemotherapy, transplantations and the treatment of other immunosuppressed patients. Using a rat model of experimental aspergillosis, the fungal siderophores ferricrocin and triacetylfusarinine C were identified as markers of aspergillosis and quantified in urine, serum and lung tissues. Biomarkers were analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization mass spectrometry using a 12T SolariX Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The limits of detection of the ferri-forms of triacetylfusarinine C and ferricrocin in the rat serum were 0.28 and 0.36 ng/mL, respectively. In the rat urine the respective limits of detection achieved 0.02 and 0.03 ng/mL. In the sera of infected animals, triacetylfusarinine C was not detected but ferricrocin concentration fluctuated in the 3-32 ng/mL range. Notably, the mean concentrations of triacetylfusarinine C and ferricrocin in the rat urine were 0.37 and 0.63 µg/mL, respectively. The MALDI FTICR mass spectrometry imaging illustrated the actual microbial ferricrocin distribution in the lung tissues and resolved the false-positive results obtained by the light microscopy and histological staining. Ferricrocin and triacetylfusarinine C detection in urine represents an innovative non-invasive indication of Aspergillus infection in a host.

Hyaluronan polymeric micelles for topical drug delivery.

Nanosized materials offer promising strategy for topical drug delivery due to their enhancing effect on drug percutaneous transport across the stratum corneum barrier. In this work, polymeric micelles made from hydrophobized hyaluronic acid (HA) were probed for skin delivery. Compared to non-polymeric micelle solutions containing similar drug amount, in vitro skin penetration analysis indicated 3 times larger deposition of drug in the epidermis and 6 times larger drug deposition in the dermis after 5h of topical treatment in Franz diffusion cells. The drug deposition was further increased with prolonged time of topical treatment. Laser confocal microscopy revealed the accumulation of both, the HA forming the vehicle and the payload, in the epidermis and dermis. Although fluorescent labeling of the HA would suggest co-transport of the HA and the drug, loading FRET pair dyes in the micellar core clearly demonstrated gradual micelle disruption with increasing skin depth. Transcellular penetration was the predominant pathway for the loaded drug. The HA polymeric micelles also demonstrated increased bioactivity of loaded compound in vitro and in vivo. In addition, the loaded micelles were found to be stable in cream formulations and thus they have great potential for topical applications for cosmetic and pharmaceutical purposes.

Pyridoindole SMe1EC2 as cognition enhancer in ageing-related cognitive decline.

Synthetic pyridoindole-type substances derived from the lead compound stobadine represent promising agents in treatment of a range of pathologies including neurological disorders. The beneficial biological effects were suggested to be likely associated with their capacity to ameliorate oxidative damage. In our study, the effect of supplementation with the derivative of stobadine, SMe1EC2, on ageing-related cognitive decline in rats was investigated. The 20-months-old male Wistar rats were administered SMe1EC2 at a low dose, 0.5 mg/kg, daily during eight weeks. Morris water maze test was performed to assess the spatial memory performances. The cell-based assays of capacity of SMe1EC2 to modulate proinflammatory generation of oxidants by microglia were also performed. The rats treated with SMe1EC2 showed significantly increased path efficiency, significantly shorter time interval of successful trials and exerted also notably lower frequencies of clockwise rotations in the pool compared to non-supplemented aged animals. Mildly improved parameters included test durations, distances to reach the platform, time in periphery of the pool and overall rotations in the water maze. However, the pyridoindole SMe1EC2 did not show profound inhibitory effect on production of nitric oxide and superoxide by activated microglial cells. In conclusion, our study suggests that pyridoindole SMe1EC2, at low doses administered chronically, can act as cognition enhancing agent in aged rats. The protective mechanism less likely involves direct modulation of proinflammatory and prooxidant state of microglia, the prominent mediators of neurotoxicity in brain ageing and neurodegeneration.

Neonatal brain injury as a consequence of insufficient cerebral oxygenation.

Neonatal brain hypoxic-ischemic injury represents a serious health care and socio-economical problem since it is one of the most common causes of mortality and morbidity of newborns. Neonatal hypoxic-ischemic encephalopathy is often associated with signs of perinatal asphyxia, with an incidence of about 2-4 per 1,000 live births and mortality rate up to 20%. In about one half of survivors, cerebral hypoxic-ischemic insult may result in more or less pronounced neuro-psychological sequelae of immediate or delayed nature, such as seizures, cerebral palsy or behavioural and learning disabilities, including attention-deficit hyperactivity disorder. Hypoxic-ischemic injury develops as a consequence of transient or permanent restriction of blood supply to the brain. Severity of hypoxic-ischemic encephalopathy varies depending on the intensity and duration of hypoxia-ischemia, on the type and size of the brain region affected, and on the maturity of the foetal/neonatal brain. Though a primary cause of hypoxic-ischemic injury is lack of oxygen in the neonatal brain, underlying mechanisms of subsequent events that are critical for developing hypoxic-ischemic encephalopathy are less understood. Their understanding is however necessary for elaborating effective management for newborns that underwent cerebral hypoxic-ischemic insult and thus are at risk of a negative outcome. The present paper summarizes current knowledge on cerebral hypoxic-ischemic injury of the neonate, fundamental processes involved in etiopathogenesis, with a special focus on cellular and molecular mechanisms and particular attention on certain controversial aspects of oxidative stress involvement.

Aspergillus infection monitored by multimodal imaging in a rat model.

Although myriads of experimental approaches have been published in the field of fungal infection diagnostics, interestingly, in 21st century there is no satisfactory early noninvasive tool for Aspergillus diagnostics with good sensitivity and specificity. In this work, we for the first time described the fungal burden in rat lungs by multimodal imaging approach. The Aspergillus infection was monitored by positron emission tomography and light microscopy employing modified Grocott's methenamine silver staining and eosin counterstaining. Laser ablation inductively coupled plasma mass spectrometry imaging has revealed a dramatic iron increase in fungi-affected areas, which can be presumably attributed to microbial siderophores. Quantitative elemental data were inferred from matrix-matched standards prepared from rat lungs. The iron, silver, and gold MS images collected with variable laser foci revealed that particularly silver or gold can be used as excellent elements useful for sensitively tracking the Aspergillus infection. The limit of detection was determined for both (107) Ag and (197) Au as 0.03 µg/g (5 µm laser focus). The selective incorporation of (107) Ag and (197) Au into fungal cell bodies and low background noise from both elements were confirmed by energy dispersive X-ray scattering utilizing the submicron lateral resolving power of scanning electron microscopy. The low limits of detection and quantitation of both gold and silver make ICP-MS imaging monitoring a viable alternative to standard optical evaluation used in current clinical settings.