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Amyloid-β42 (Aβ42) peptides are central to the amyloid pathology in Alzheimer's disease (AD). As biological mimetics, properties of synthetic Aβ peptides usually vary between vendors and batches, thus impacting the reproducibility of experimental studies. Here, we tested recombinantly expressed Aβ42 (Asp1 to Ala42) against synthetic Aβ42 from different suppliers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), circular dichroism (CD) spectroscopy, thioflavin T aggregation, surface plasmon resonance, and MTT cell viability assays. Overall, our recombinant Aβ42 provided a reproducible mimetic of desired properties. Across experimental approaches, the combined detection of Aβ42 dimers and random coil to β-sheet transition only correlated with aggregation-prone and cytotoxic peptides. Conclusively, combining MALDI-MS with CD appears to provide a rapid, reliable means to predict the 'bioactivity' of Aβ42.

The purpose of the current study is to uncover the impact of small liganded gold nanoclusters with 10 gold atoms and 10 glutathione ligands (Au

We have previously developed a unique 8-amino acid Aβ42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aβ42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aβ42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aβ42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease.

The purpose of the current study is to uncover the impact of small liganded gold nanoclusters with 10 gold atoms and 10 glutathione ligands (Au10SG10) on several biomarkers in human microglia. We established the links connecting the atomically precise structure of Au10SG10 with their properties and changes in several biomolecules under oxidative stress. Au10SG10 caused the loss of mitochondrial metabolic activity, increased lipid peroxidation and translocation of an alarmin molecule, high mobility group box 1 (HMGB1), from the nucleus to the cytosol. Molecular modeling provided an insight into the location of amino acid interaction sites with Au10SG10 and the nature of bonds participating in these interactions. We show that Au10SG10 can bind directly to the defined sites of reduced, oxidized, and acetylated HMGB1. Further studies with similar complementary approaches merging live-cell analyses, determination of biomarkers, and cell functions could lead to optimized gold nanoclusters best suited for diagnostic and bioimaging purposes in neuroscience.

Soluble oligomers of the 42-amino acid amyloid beta (Aβ42) peptide are highly toxic and suspected as the causative agent of synaptic dysfunction and neuronal loss in Alzheimer's disease (AD). Previously, we have shown that a small, D-amino acid Aβ42-oligomer interacting peptide (D-AIP) can neutralize human Aβ42-mediated toxicity using in vitro and cell-based assays. In the present longitudinal study using a transgenic Drosophila melanogaster model, advanced live confocal imaging and mass spectrometry imaging (MALDI-MSI) showed that the eight amino acid D-AIP can attenuate Aβ42-induced toxicity in vivo. By separating male and female flies into distinct groups, the resultant distribution of ingested D-AIP was different between the sexes. The Aβ42-induced 'rough eye' phenotype could be rescued in the female transgenics, likely because of the co-localization of D-AIP with human Aβ42 in the female fly heads. Interestingly, the phenotype could not be rescued in the male transgenics, likely because of the co-localization of D-AIP with a confounding male-specific sex peptide (Acp70A candidate in MSI spectra) in the gut of the male flies. As a novel, more cost-effective strategy to prevent toxic amyloid formation during the early stages of AD (i.e. neutralization of toxic low-order Aβ42 oligomers without creating larger aggregates in the process), our longitudinal study establishes that D-AIP is a stable and highly effective neutralizer of toxic Aβ42 peptides in vivo. Cover Image for this issue: doi: 10.1111/jnc.14512.

There is increasing evidence suggesting that amyloidogenic proteins might form deposits in non-neuronal tissues in neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. However, the detection of these aggregation-prone proteins within the human skin has been controversial. Using immunohistochemistry (IHC) and mass spectrometry tissue imaging (MALDI-MSI), fresh frozen human skin samples were analyzed for the expression and localization of neurodegenerative disease-related proteins. While α-synuclein was detected throughout the epidermal layer of the auricular samples (IHC and MALDI-MSI), tau and Aβ34 were also localized to the epidermal layer (IHC). In addition to Aβ peptides of varying length (e.g., Aβ40, Aβ42, Aβ34), we also were able to detect inflammatory markers within the same sample sets (e.g., thymosin β-4, psoriasin). While previous literature has described α-synuclein in the nucleus of neurons (e.g., Parkinson's disease), our current detection of α-synuclein in the nucleus of skin cells is novel. Imaging of α-synuclein or tau revealed that their presence was similar between the young and old samples in our present study. Future work may reveal differences relevant for diagnosis between these proteins at the molecular level (e.g., age-dependent post-translational modifications). Our novel detection of Aβ34 in human skin suggests that, just like in the brain, it may represent a stable intermediate of the Aβ40 and Aβ42 degradation pathway.

Aim: To enhance the drug delivery to the brain with an oil-in-water nanoemulsion of pretomanid via intranasal (IN) administration. Materials & methods: The study involved 70 male Sprague–Dawley rats (160–180 g) that received either 20 mg/kg body weight (b.w.) a nanoemulsion or a 20 mg/kg b.w. of pretomanid in solution via the IN route. The drug was quantified by liquid chromatography–tandem mass spectrometry to investigate whole tissue–drug concentrations, and mass spectrometric imaging to visualize drug localization in the brain. Results: Nanoemulsion delivery concentrations of pretomanid in the brain reached peak concentrations (Cmax) of 12,062.3 ng/g that is significantly higher than the required therapeutic level. The mass spectrometric imaging analysis clearly showed a time dependent and uniform distribution in the brain. Conclusion: The results of this study show that IN delivery of oil-in-water nanoemulsion may be very promising for targeting anatomical tuberculosis reservoirs, such as the brain.

A wide range of microorganisms can infect the central nervous system (CNS). The immune response of the CNS provides limited protection against microbes penetrating the blood-brain barrier. This results in a neurological deficit and sometimes leads to high morbidity and mortality rates despite advanced therapies. For the last two decades, different studies have expanded our understanding of the molecular basis of human neuroinfectious diseases, especially concerning the contributions of mast cell interactions with other central nervous system compartments. Brain mast cells are multifunctional cells derived from the bone marrow and reside in the brain. Their proximity to blood vessels, their role as "first responders" their unique receptors systems and their ability to rapidly release pathogen responsive mediators enable them to exert a crucial defensive role in the host-defense system. This review describes key biological and physiological functions of mast cells, concerning their ability to recognize pathogens via various receptor systems, followed by a coordinated and selective mediator release upon specific interactions with pathogenic stimulating factors. The goal of this review is to direct attention to the possibilities for therapeutic applications of mast cells against bacterial and viral related infections. We also focus on opportunities for future research activating mast cells via adjuvants.

Alzheimer's disease (AD) is characterized by extracellular deposition of amyloid-β (Aβ) plaques. These protein deposits impair synaptic plasticity thereby producing a progressive decline in cognitive function. Current therapies are merely palliative and only slow cognitive decline. Poly-N-methylated Aβ-Peptide C-Terminal Fragments (MEPTIDES) were recently shown to reduce Aβ toxicity in vitro and in Drosophila melanogaster, however whether these novel compounds are effective in inhibiting Aβ-induced toxicity in the mammalian brain remains unclear. We therefore investigated whether MEPTIDES have the ability to reduce the neurotoxic effects of Aβ in male Sprague-Dawley (SD) rats. Aβ42 (100 μg, 2 mM) or vehicle (0.15 M Tris buffer) was stereotaxically injected bilaterally into the dorsal hippocampus at a rate of 1 μl/min for 10 min. The effects on hippocampal-mediated learning were subsequently assessed using the Morris water maze (MWM). The presence of apoptotic activity was also assessed by determining the expression levels of active caspase-3 using real-time polymerase chain reaction and Western Blot techniques. In addition, half of the animals (n = 20) received an intraperitoneal (i.p.) injection of MEPTIDES (2 mg/kg) 48 h after intrahippocampal injection of Aβ42. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI -TOF) mass spectrometry (MS) showed that MEPTIDES crossed the blood brain barrier (BBB) and revealed their distribution in the rat brain. Rats treated with Aβ42 displayed spatial learning deficits and increased hippocampal caspase-3 gene (CASP-3) expression which was reversed by subsequent injection of MEPTIDES. The present results show that MEPTIDES have the potential to reverse the toxic effects of Aβ42 in vivo.

Additional file 4. Cerebral Amyloid Angiopathy (CAA) quantification.

Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra-abdominal infections. A selective, accurate and reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel-Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C18 column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150-1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time-points. Copyright © 2015 John Wiley & Sons, Ltd.

This is the first report of clofazimine (CFZ) penetration and distribution in normal mouse brain. Mice were administered 25 mg/kg CFZ or 100 mg/kg CFZ orally, daily for 2 weeks. Animals were sacrificed and blood and brain tissues were harvested. Liquid chromatography tandem mass spectrometry showed high concentrations of CFZ in homogenized brain, with 100 mg/kg dose having significantly higher concentration than 25 mg/kg. Matrix-assisted laser desorption/ionization spectrometric imaging of brain sections showed widespread tissue distribution of CFZ. Our results show dose dependent localization in brain.

1. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) combines the sensitivity and selectivity of mass spectrometry with spatial analysis to provide a new dimension for histological analyses of the distribution of drugs in tissue. Pretomanid is a pro-drug belonging to a class of antibiotics known as nitroimidizoles, which have been proven to be active under hypoxic conditions and to the best of our knowledge there have been no studies investigating the distribution and localisation of this class of compounds in the brain using MALDI MSI. 2. Herein, we report on the distribution of pretomanid in the healthy rat brain after intraperitoneal administration (20 mg/kg) using MALDI MSI. Our findings showed that the drug localises in specific compartments of the rat brain viz. the corpus callosum, a dense network of neurons connecting left and right cerebral hemispheres. 3. This study proves that MALDI MSI technique has great potential for mapping the pretomanid distribution in uninfected tissue samples, without the need for molecular labelling.

A selective, sensitive and high performance liquid chromatography-tandem mass spectrometry (LC–(ESI)MS/MS) method has been developed and validated for the quantification of the potent antitubercular drug candidate, PA-824, in rat plasma, lung and brain tissues. Sample clean-up involved protein precipitation and solid-phase extraction. Chromatographic separation was performed on YMC Triart C18 column (150 mm × 3.0 mm, 3.0 μm). The method was validated over the concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. Evaluation of the pharmacokinetic properties of PA-824 utilized Sprague Dawley rats with a dosage of 20 mg/kg at various time points. The new method was applied successfully for the determination of PA-824 with liquid desorption followed by liquid chromatography with ultra-high resolution quadrupole time-of-flight mass spectrometry in the different biological samples.

The method of direct mass spectrometry profiling is reliable and reproducible for the rapid identification of clinical isolates of bacteria and fungi. This is the first study evaluating the approach of MALDI-TOF mass spectrometry profiling for rapid identification of carbapenemase-resistant enterobacteriaceae (CRE). Proof of concept was achieved by the discrimination of CRE using MALDI Biotyper MS based on the protein. This profiling appears promising by the visual observation of consist- ent unique peaks, albeit low intensity, that could be picked up from the mean spectra (MSP) method. The Biotyper MSP creation and identification methods needed to be optimized to provide significantly improved differences in scores to allow for subspe- cies identification with and without carbapenemases. These spectra were subjected to visual peak picking and in all cases; there were pertinent differences in the presence or absence of potential biomarker peaks to differentiate isolates. We also evaluated this method for potential discrimination between different carbapenemases bacteria, utilizing the same strategy. Based on our data and pending further investigation in other CREs, MALDI-TOF MS has potential as a diagnostic tool for the rapid identification of even closely related carbapenemases but would require a paradigm shift in which Biotyper suppliers enable more flexible software control of mass spectral profiling methods.

1. TBA-354 was a promising antitubercular compound with activity against both replicating and static Mycobacterium tuberculosis (M.tb), making it the focal point of many clinical trials conducted by the TB Alliance. However, findings from these trials have shown that TBA-354 results in mild signs of reversible neurotoxicity; this left the TB Alliance with no other choice but to stop the research. 2. In this study, mass spectrometric methods were used to evaluate the pharmacokinetics and spatial distribution of TBA-354 in the brain using a validated liquid chromatography tandem-mass spectrometry (LCMS/MS) and mass spectrometric imaging (MSI), respectively. Healthy female Sprague-Dawley rats received intraperitoneal (i.p.) doses of TBA-354 (20 mg/kg bw). 3. The concentrationtime profiles showed a gradual absorption and tissue penetration of TBA-354 reaching the Cmax at 6 h post dose, followed by a rapid elimination. MSI analysis showed a time-dependent drug distribution, with highest drug concentration mainly in the neocortical regions of the brain. 4. The distribution of TBA-354 provides a possible explanation for the motor dysfunction observed in clinical trials. These results prove the importance of MSI as a potential tool in preclinical evaluations of suspected neurotoxic compounds.

A study was undertaken to determine the neuroprotective potential of Linezolid (LIN) in an animal model. Female Sprague–Dawley rats were either given a single (100 mg/kg) dose or treated daily for 4 weeks. A validated LC–MS/MS method was used to measure LIN levels in plasma and brain, this was paired with mass spectrometry imaging to determine the tissue spatial distribution of the drug. The results showed that after a single dose there was poor penetration of the drug into the brain. With multiple doses there were high tissue levels, with the drug reaching steady state in subsequent weeks. LIN displayed a promising distribution pattern with localisation in the brainstem. Systemic circulation is fed into the brain by the carotid and vertebral arteries which enter through the brain stem, therefore high drug concentrations is this area may protect against infectious agents entering via this route.

Given the recent explosion of mass spectrometric imaging (MSI), it has become easier to assess drug tissue localisation without the use of radiolabeling and other more complex methods (such as PET and MRI). For MSI tissue preparation is of utmost importance, however, the lung in particular does pose some difficulties with imaging since it is made up of a number of air-filled alveoli. These organs are known to collapse when the thoracic cavity is pierced, losing its structural integrity and giving poor histological representation for drug distribution analysis. The use of cryoprotectants as a tissue inflation media will aid in the preservation of the lung's structural integrity during MSI experiments involving small molecule distribution. Various established cryoprotectants (DMSO, PvP, ethylene glycol, sucrose, DMEM, control serum, OCT) were selected as lung inflation media for MSI analysis of gatifloxacin (GAT). Female Sprague-Dawley rats were treated with GAT (10 mg/kg b.w) via i.p. injection. After 15 min the animals were terminated by halothane overdose, and each set of tissue inflated with a specific agent. Cryosections were made and MSI conducted to determine drug tissue distribution. During the early stages of the experimental procedure some crypreservatives were eliminated due to difficulties with sample preparation. While others displayed excellent preservation of the tissue structure and integrity. Following MSI analysis, some agents showed homogenous drug distribution while some displayed heterogeneous distribution favoring the basal periphery. Taking into account the physiology of the lung and previous MRI investigations of its perfusion, it is expected that a systemically administered drug would localize in the basal areas. DMSO and DMEM proved to display this distribution pattern while keeping structural integrity intact. However, the later was ruled out since it showed complete suppression of GAT in solution. From the cryoprotectants selected for this study, DMSO is the most promising lung inflation media focusing on small molecule distribution via MSI.

1. The penetration of tetracyclines into the brain has been widely documented. The aim of this work was to develop a matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI MSI) method for the molecular histology of doxycycline (DOX) in the healthy rat brain. 2. The time-dependent distribution was investigated after an i.p. dose of 25 mg/kg at 0, 5, 30, 120, 240, 360 and 480 min postdose. LCMS/MS was used to quantify the drug in plasma and brain homogenates and MALDI MSI was used to determine the distribution of the analyte. 3. Within the first-hour postdose, the drug showed slow accumulation into the plasma and brain tissues. DOX brain concentration gradually increased and reached a peak (Cmax) of 1034.9 ng/mL at 240 min postdose, resulting in a brain plasma ratio of 31%. The images acquired by MSI matched the quantification results and clearly showed drug distribution over the entire rat brain coronal section from 5 min and its slow elimination after 360-min postdose. 4. Our findings confirm that MALDI MSI provides an advanced, label-free and faster alternative technique for xenobiotic distribution such as DOX in tissues, making it an essential drug discovery tool for other possible neuroprotective agents.

Fluoroquinolones are broad-spectrum antibiotics with efficacy against a wide range of pathogenic microbes associated with respiratory and meningeal infections. The potential toxicity of this class of chemical agents is a source of major concern and is becoming a global issue. The aim of this study was to develop a method for the brain distribution and the pharmacokinetic profile of gatifloxacin in healthy Sprague-Dawley rats, via Multicenter matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed a sensitive LC-MS/MS method to quantify gatifloxacin in plasma, lung, and brain homogenates. A pharmacokinetic profile was observed where there is a double peak pattern; a sharp initial increase in the concentration soon after dosing followed by a steady decline until another increase in concentration after a longer period post dosing in all three biological samples was observed. The imaging results showed the drug gradually entering the brain via the blood brain barrier and into the cortical regions from 15 to 240 min post dose. As time elapses, the drug leaves the brain following the same path as it followed on its entry and finally concentrates at the cortex. Copyright © 2015 John WileySons, Ltd.

Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra-abdominal infections. A selective, accurate and reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel-Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C18 column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150-1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time-points. Copyright © 2015 John WileySons, Ltd.

Lansoprazole (LPZ) is a commercially available proton-pump inhibitor whose primary metabolite, lansoprazole sulfide (LPZS) was recently reported to have in vitro and in vivo activity against Mycobacterium tuberculosis. It was also reported that a 300 mg kg-1 oral administration of LPZS was necessary to reach therapeutic levels in the lung, with the equivalent human dose being unrealistic. A validated liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for the simultaneous quantification LPZ and LPZS in rat plasma and lung homogenates was developed. We administered 15 mg kg-1 oral doses of LPZ to a healthy rat model to determine the pharmacokinetics of its active metabolite, LPZS, in plasma and lung tissue. We found that the LPZS was present in amounts that were below the limit of quantification. This prompted us to administer the same dose of LPZS to the experimental animals intraperitoneally (i.p.). Using this approach, we found high concentrations of LPZS in plasma and lung, 7841.1 and 9761.2 ng mL-1 , respectively, which were significantly greater than the minimum inhibitory concentration (MIC) for Mycobacterium tuberculosis. While oral and i.p. administration of LPZ resulted in significant concentrations in the lung, it did not undergo sufficient cellular conversion to its anti-TB metabolite. However, when LPZS itself was administered i.p., significant amounts penetrated the tissue. These results have implications for future in vivo studies exploring the potential of LPZS as an anti-TB compound.

Additional file 1. Primary antibodies used in immunofluorescence (IF), immunocytochemistry (ICC) and western blot (WB).

Additional file 2. Additional Aβ34-PDGFR-β immunostainings. ThioS staining. Vessel density quantification.

Additional file 3. Additional in vitro pericyte culture results.

Additional file 1. Primary antibodies used in immunofluorescence (IF), immunocytochemistry (ICC) and western blot (WB).

Additional file 2. Additional Aβ34-PDGFR-β immunostainings. ThioS staining. Vessel density quantification.

Additional file 3. Additional in vitro pericyte culture results.