Chromatographic Separation and Quantitation of Sphingolipids from the Central Nervous System or Any Other Biological Tissue.

Chromatographic separation and purification of an individual lipid to homogeneity have long been introduced. Using this concept, a more precise method has been developed to identify and characterize the sphingolipid composition(s) using a small amount (30 mg) of biological sample. Sphingolipids (lipids containing sphingosine or dihydrosphingosine) are well-known regulators of the central nervous system development and play a critical role in neurodegenerative diseases. Introducing a silicic acid column chromatography, sphingolipid components have been separated to individual fractions such as ceramide, glucosyl/galactosylceramide, other neutral and acidic glycosphingolipids, including (dihydro)sphingosine and psychosine; as well as phospholipids from which individual components are quantified employing a single or combination of other advanced chromatography procedures such as thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography-mass spectrometry.

An ultrahigh-performance liquid chromatography-tandem mass spectrometry method for quantification of methotrexate and 7-hydroxy-methotrexate and application for therapeutic drug monitoring in patients with central nervous system lymphoma.

A method using ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed, validated, and applied to simultaneously determine plasma methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) in 117 patients with central nervous system (CNS) lymphoma. The ion transitions utilized were m/z 455.2 > 308.2 for MTX and m/z 471.2 > 324.1 for 7-OH-MTX. Samples were prepared through protein precipitation using methanol. Chromatographic separation was achieved within 3.0 min on a CMS9030 column (Ruixi, 2.1 × 50 mm, 3 µm) through a gradient elution of methanol and a 10% ammonium acetate solution at a flow rate of 0.4 mL/min. The method demonstrated linearity in the concentration range of 0.05-10 µM for MTX and 0.25-50 µM for 7-OH-MTX. The intra- and inter-day inaccuracy ranged from -7.38% to 7.83%, and the imprecision was less than 6.00% for both analytes. The recovery and matrix effect normalized by the internal standard (MTX-D3 ) remained consistent. Both analytes remained stable under nine different storage conditions. In patients with CNS lymphoma, MTX levels at 12 h and 7-OH-MTX levels at 12, 36, and 60 h after dosing in individuals with impaired renal function were significantly higher compared with those with normal renal function. 7-OH-MTX could potentially serve as a superior indicator for nephrotoxicity compared with MTX.

Oncogene-induced senescence in meningiomas-an immunohistochemical study.

PURPOSE: Meningiomas are tumours originating from meningothelial cells, the majority belonging to grade 1 according to the World Health Organization classification of the tumours of the Central Nervous System. Factors contributing to the progression to the higher grades (grades 2 and 3) have not been elucidated yet. Senescence has been proposed as a potential mechanism constraining the malignant transformation of tumours. Senescence-associated beta-galactosidase (SA-ß-GAL) and inhibitors of cyclin-dependent kinases p16 and p21 have been suggested as senescence markers. METHODS: We analysed 318 meningiomas of total 343 (178 grade 1, 133 grade 2 and 7 grade 3). Tissue microarrays were constructed and stained immunohistochemically, using antibodies for SA-ß-GAL, p16 and p21. RESULTS: The positive correlation of the tumour grade with the expression of p16 (p = 0.016) and SA-ß-GAL (p = 0.002) was observed. The expression of p16 and SA-ß-GAL was significantly higher in meningiomas grade 2 compared to meningiomas grade 1 (p = 0.006 and p = 0.004, respectively). SA-ß-GAL positivity positively correlated with p16 and p21 in the whole cohort. In grade 2 meningiomas, a positive correlation was only between SA-ß-GAL and p16. Correlations of senescence markers in meningiomas grade 2 were not present. CONCLUSION: Our findings suggest the senescence activation in meningiomas grade 2 as a potential mechanism for the restraining of tumour growth and give hope for applying of promising senolytic therapy.

Liquid biopsy in the setting of leptomeningeal metastases: a systematic review and meta-analysis.

PURPOSE: The blood-brain barrier can prevent circulating tumor DNA (ctDNA) derived from the central nervous system from entering the blood making it challenging to evaluate molecular features of leptomeningeal metastasis (LM). Accordingly, we sought to systematically compare the diagnostic power or significance of ctDNA derived from cerebrospinal fluid (CSF) compared to plasma ctDNA in patients with LM. METHODS: A systematic review and meta-analysis was performed under the PRISMA guideline. We used PubMed, EMBASE, and the EuroPMC to search the literature using combinations of the following terms: circulating tumor DNA, ctDNA, circulating tumor cell, brain metastasis, leptomeningeal metastasis, outcome(s), and prognosis. We included all available English language studies that compared the diagnostic significance of CSF derived and serum ctDNA. All eligible studies level of bias was assessed using the New Castle Ottawa Scale (NOS). RESULTS: Our meta-analysis from 6 included studies (n = 226) that confirmed the diagnostic power of liquid biopsies in detecting genomic alteration is better when taking a CSF-derived samples than from the plasma (RR 1.46 [0.93; 2.29]; I2 = 92%; p-value < 0.01). CONCLUSION: CSF ctDNA is better at describing molecular landscape for LM; such an understanding may ultimately help inform patient treatment and responses to therapy.

Distribution, cellular localization, and colocalization of several peptide neurotransmitters in the central nervous system of Aplysia.

Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk Aplysia, with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult Aplysia with antisera raised against the Aplysia peptide FMRFamide and two mammalian peptides that are also found in Aplysia, cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the Aplysia nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as XSOM and XPHI There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for XSOM and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of Aplysia is advantageous for further testing these ideas.

Unusual Toxoplasma infection of the eye and central nervous system in an HIV-positive patient.

OBJECTIVE: To report on a unique combination of cerebral toxoplasmosis and ocular toxoplasmosis in an HIV-positive patient in Slovakia. METHODS: A 35-year-old heterosexual patient who presented with headache and major seizures underwent computed tomography (CT) and magnetic resonance imaging (MRI). Based on clinical findings, serological tests for toxoplasmosis were performed on serum and ocular fluid specimens. PCR was also used to detect Toxoplasma gondii and cytomegalovirus DNA. Goldmann and Witmer coefficient calculation was applied to demonstrate the synthesis of intraocular IgG antibodies. RESULTS: CT and MRI revealed cystic lesions suspected of metastasis in the occipital and temporal regions, and we searched for the primary tumor. After vision loss in the left eye, which rapidly progressed to complete blindness, an eye examination detected macular edema. Anti-edema treatment was initiated. HIV positivity with a very low CD4 T-cell count (20/μL) was found, and the viral load was 100 400 HIV-RNA copies/ml. The serum was positive for anti-Toxoplasma IgG antibodies (> 200 IU/mL), IgM negative, and IgA borderline. As toxoplasmic encephalitis and retinitis were suspected, antitoxoplasmic therapy with pyrimethamine, spiramycin, and folinic acid was started. The ophthalmologist considered cytomegalovirus retinitis, which was not confirmed by serology or PCR. In contrast, the presence of IgG antibodies in ocular fluid and serum with the calculation of the Goldmann-Witmer coefficient (GW = 32) as well as PCR DNA positivity pointed to Toxoplasma gondii as the etiological agent. Follow-up MRI scan confirmed regression of the pathological lesions, neurological deficit also improved, CD4 T-lymphocytes increased above 200/μL, but blindness of the left eye persisted. CONCLUSION: CT and MRI scans offered no clue as to Toxoplasma etiology of the brain and eye involvement in an HIV-positive patient, which was only confirmed by laboratory tests. Due to the delay in the diagnosis of toxoplasmosis, time from the epileptic seizure to treatment initiation was 16 days, which assumedly caused irreversible blindness in the patient.

Central nervous system sarcoma with ATXN1::DUX4 fusion expands the concept of CIC-rearranged sarcoma.

CIC-rearranged sarcoma is a high-grade sarcoma, most often harboring CIC::DUX4 fusion, and is characterized by a distinct round cell histology, co-expression of ETV4 and WT1, and a specific DNA methylation class. Herein, we report a brain tumor with ATXN1::DUX4 that had an indistinguishable phenotype and DNA methylation profile from CIC-rearranged sarcoma. A 40-year-old man presented with a 5 cm hemorrhagic mass in the right frontal lobe of the cerebrum. The tumor was resected and histologically showed a dense proliferation of relatively monomorphic round cells with multifocal myxoid changes. Immunohistochemically, the tumor was diffusely positive for ETV4, WT1, and DUX4. Through classic histomorphology and immunoprofile, the tumor was provisionally diagnosed as CIC-rearranged sarcoma. However, no CIC fusions or mutations were identified using CIC break-apart fluorescence in situ hybridization (FISH) or FoundationOne CDx. Despite multiple surgeries and adjuvant chemoradiation therapy, the patient succumbed 16 months after presentation. RNA exome sequencing detected an in-frame intraexonic ATXN1 (exon 9)::DUX4 (exon 1) fusion, which was validated by reverse transcription-polymerase chain reaction and ATXN1 FISH assay. Upon DNA methylation analysis, the tumor matched with CIC-rearranged sarcoma both by the Deutsche Krebsforschungszentrum classifier and t-distributed stochastic neighbor embedding. Along with a recent report of a similar pediatric brain tumor, the present case suggests that ATXN1::DUX4 is a recurrent alternative molecular event in the sarcoma type that is presently defined by CIC rearrangement, which prompts an expansion of the tumor concept.

Truly-Biocompatible Gold Catalysis Enables Vivo-Orthogonal Intra-CNS Release of Anxiolytics.

Being recognized as the best-tolerated of all metals, the catalytic potential of gold (Au) has thus far been hindered by the ubiquitous presence of thiols in organisms. Herein we report the development of a truly-catalytic Au-polymer composite by assembling ultrasmall Au-nanoparticles at the protein-repelling outer layer of a co-polymer scaffold via electrostatic loading. Illustrating the in vivo-compatibility of the novel catalysts, we show their capacity to uncage the anxiolytic agent fluoxetine at the central nervous system (CNS) of developing zebrafish, influencing their swim pattern. This bioorthogonal strategy has enabled -for the first time- modification of cognitive activity by releasing a neuroactive agent directly in the brain of an animal.

Neural Cell Membrane-Coated Nanoparticles for Targeted and Enhanced Uptake by Central Nervous System Cells.

Targeted drug delivery to specific neural cells within the central nervous system (CNS) plays important roles in treating neurological disorders, such as neurodegenerative (e.g., targeting neurons) and demyelinating diseases [e.g., targeting oligodendrocytes (OLs)]. However, the presence of many other cell types within the CNS, such as microglial and astrocytes, may lead to nonspecific uptake and subsequent side effects. As such, exploring an effective and targeted drug delivery system is of great necessity. Synthetic micro-/nanoparticles that have been coated with biologically derived cellular membranes have emerged as a new class of drug delivery vehicles. However, the use of neural cell-derived membrane coatings remains unexplored. Here, we utilized this technique and demonstrated the efficacy of targeted delivery by using four types of cell membranes that were derived from the CNS, namely, microglial, astrocytes, oligodendrocyte progenitor cells (OPCs), and cortical neurons. A successful cell membrane coating over poly(ε-caprolactone) nanoparticles (NPs) was confirmed using dynamic light scattering, zeta potential measurements, and transmission electron microscopy. Subsequently, an extensive screening of these cell membrane-coated NPs was carried out on various CNS cells. Results suggested that microglial and OLs were the most sensitive cell types toward cell membrane-coated NPs. Specifically, cell membrane-coated NPs significantly enhanced the uptake efficiency of OLs (p < 0.001). Additionally, a temporal uptake study indicated that the OLs took up microglial membrane-coated NPs (DPP-PCL-M Mem) most efficiently. Besides that, coating the NPs with four types of the CNS cell membrane did not result in obvious specific uptake in microglial but reduced the activation of microglial, especially for DPP-PCL-M Mem (p < 0.01). Taken together, DPP-PCL-M Mem were uptaken most efficiently in OLs and did not induce significant microglial activation and may be most suitable for CNS drug delivery applications.

A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system.

Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques' CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain.

Potential Genetic Contributions of the Central Nervous System to a Predisposition to Elite Athletic Traits: State-of-the-Art and Future Perspectives.

Recent remarkable advances in genetic technologies have allowed for the identification of genetic factors potentially related to a predisposition to elite athletic performance. Most of these genetic variants seem to be implicated in musculoskeletal and cardiopulmonary functions. Conversely, it remains unclear whether functions of the central nervous system (CNS) genetically contribute to elite athletic traits, although the CNS plays critical roles in exercise performance. Accumulating evidence has highlighted the emerging implications of CNS-related genes in the modulation of brain activities, including mental performance and motor-related traits, thereby potentially contributing to high levels of exercise performance. In this review, recent advances are summarized, and future research directions are discussed in regard to CNS-related genes with potential roles in a predisposition to elite athletic traits.

A mechanistic view on the neurotoxic effects of air pollution on central nervous system: risk for autism and neurodegenerative diseases.

Many reports have shown a strong association between exposure to neurotoxic air pollutants like heavy metal and particulate matter (PM) as an active participant and neurological disorders. While the effects of these toxic pollutants on cardiopulmonary morbidity have principally been studied, growing evidence has shown that exposure to polluted air is associated with memory impairment, communication deficits, and anxiety/depression among all ages. So, these toxic pollutants in the environment increase the risk of neurodegenerative disease, ischemia, and autism spectrum disorders (ASD). The precise mechanisms in which air pollutants lead to communicative inability, social inability, and declined cognition have remained unknown. Various animal model studies show that amyloid precursor protein (APP), processing, oxidant/antioxidant balance, and inflammation pathways change following the exposure to constituents of polluted air. In the present review study, we collect the probable molecular mechanisms of deleterious CNS effects in response to various air pollutants.

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration.

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

Insect Body Defence Reactions against Bee Venom: Do Adipokinetic Hormones Play a Role?

Bees originally developed their stinging apparatus and venom against members of their own species from other hives or against predatory insects. Nevertheless, the biological and biochemical response of arthropods to bee venom is not well studied. Thus, in this study, the physiological responses of a model insect species (American cockroach, Periplaneta americana) to honeybee venom were investigated. Bee venom toxins elicited severe stress (LD50 = 1.063 uL venom) resulting in a significant increase in adipokinetic hormones (AKHs) in the cockroach central nervous system and haemolymph. Venom treatment induced a large destruction of muscle cell ultrastructure, especially myofibrils and sarcomeres. Interestingly, co-application of venom with cockroach Peram-CAH-II AKH eliminated this effect. Envenomation modulated the levels of carbohydrates, lipids, and proteins in the haemolymph and the activity of digestive amylases, lipases, and proteases in the midgut. Bee venom significantly reduced vitellogenin levels in females. Dopamine and glutathione (GSH and GSSG) insignificantly increased after venom treatment. However, dopamine levels significantly increased after Peram-CAH-II application and after co-application with bee venom, while GSH and GSSG levels immediately increased after co-application. The results suggest a general reaction of the cockroach body to bee venom and at least a partial involvement of AKHs.

Basic quantitative morphological methods applied to the central nervous system.

Generating numbers has become an almost inevitable task associated with studies of the morphology of the nervous system. Numbers serve a desire for clarity and objectivity in the presentation of results and are a prerequisite for the statistical evaluation of experimental outcomes. Clarity, objectivity, and statistics make demands on the quality of the numbers that are not met by many methods. This review provides a refresher of problems associated with generating numbers that describe the nervous system in terms of the volumes, surfaces, lengths, and numbers of its components. An important aim is to provide comprehensible descriptions of the methods that address these problems. Collectively known as design-based stereology, these methods share two features critical to their application. First, they are firmly based in mathematics and its proofs. Second and critically underemphasized, an understanding of their mathematical background is not necessary for their informed and productive application. Understanding and applying estimators of volume, surface, length or number does not require more of an organizational mastermind than an immunohistochemical protocol. And when it comes to calculations, square roots are the gravest challenges to overcome. Sampling strategies that are combined with stereological probes are efficient and allow a rational assessment if the numbers that have been generated are "good enough." Much may be unfamiliar, but very little is difficult. These methods can no longer be scapegoats for discrepant results but faithfully produce numbers on the material that is assessed. They also faithfully reflect problems that associated with the histological material and the anatomically informed decisions needed to generate numbers that are not only valid in theory. It is within reach to generate practically useful numbers that must integrate with qualitative knowledge to understand the function of neural systems.

Putative role of vitamin D in the mechanism of alcoholism and other addictions - a hypothesis.

OBJECTIVE: Vitamin D deficiency may be a clinical problem in patients with addictions. The authors systematically searched for studies addressing vitamin D and addiction and develop a hypothesis which can direct future research of the possible mechanistic role of vitamin D in the process of addiction. METHODS: Systematic review of the literature found in PubMed and EMBASE followed by narrative review combined with clinical experiences leading to hypotheses for future research. RESULTS: Only five articles were identified about a role of vitamin D in the pathophysiology of addiction. Their results are in line with a possible influence of vitamin D in dopaminergic transmission. The cerebral vitamin D status depends on the functionality of genetic variants of vitamin D receptor and other involved genes. Routine serum calcidiol levels may not adequately reflect cerebral vitamin D status. Uncertainty exists regarding appropriate calcidiol blood levels and proper dosages for affecting the central nervous system (CNS). CONCLUSIONS: The putative pathophysiological role of vitamin D in substance abuse has been insufficiently studied which calls to more studies how to measure cerebral vitamin D status in clinical practice. Research is indicated whether vitamin D supplementation should use higher dosages and aim to reach higher calcidiol serum levels. Measuring dopaminergic functioning within the prefrontal cortex as reflected by neuropsychological tests selected as suitable could be a appropriate proxy for the cerebral vitamin D status when studying the pharmacogenomics of this functionality in patients.

Establishment of an in Vitro Human Blood-Brain Barrier Model Derived from Induced Pluripotent Stem Cells and Comparison to a Porcine Cell-Based System.

The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ω∙cm2 and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs).

Assessment of Toxicity of Nanoparticles Using Insects as Biological Models.

Nanomaterials have become increasingly important in medicine, manufacturing, and consumer products. A fundamental understanding of the effects of nanoparticles (NPs) and their interactions with biomolecules and organismal systems has yet to be achieved. In this chapter, we firstly provide a brief review of the interactions between nanoparticles and biological systems. We then provide an example by describing a novel method to assess the effects of NPs on biological systems, using insects as a model. Nanoparticles were injected into the central nervous system of the discoid cockroach (Blaberus discoidalis). It was found that insects became hyperactive compared to negative control (water injections). Our method could provide a generic method of assessing nanoparticles toxicity.

Anthocyanins: Plant Pigments, Food Ingredients or Therapeutic Agents for the CNS? A Mini-Review Focused on Clinical Trials.

Anthocyanins are plant pigments present in flowers, leaves or fruits with polyphenolic structure belonging to the group of flavonoids. From a nutritional approach, they are the most abundant flavonoids in dietary sources responsible for the antioxidant properties of those foods. In addition, the food and pharma industries have used anthocyanins as food additives or excipients due to its colorant properties. However, beyond its antioxidant effects, anthocyanins may also act as therapeutic agents due to neuroprotective, antidiabetic and cardioprotective properties. There is a growing body of evidence suggesting the role of these compounds in the Central Nervous System (CNS). Previous in vitro and animal studies have suggested neuroprotective benefits, but we here review human interventions made with anthocyanins in relation to cognition, insomnia, anxiety or depression. Due to the link between oxidative stress and neurodegenerative disorders, human studies conducted on healthy volunteers evaluating oxidative stress parameters have also been included. As reviewed here, very few human studies (only ten) have been performed in the area of CNS; however, considering the obtained outcomes in those trials together with human interventions in relation with oxidative stress as well as data showing neuroprotective effects from preclinical experiments, we suggest that anthocyanins may have potential benefits for the CNS.

Differential Post-Translational Amino Acid Isomerization Found among Neuropeptides in Aplysia californica.

d-Amino acid-containing peptides (DAACPs) make up a class of post-translationally modified peptides in animals that play important roles as cell-to-cell signaling molecules. Despite the functional importance of l- to d-residue isomerization, little is known about its prevalence, mostly due to difficulties associated with detecting differences in peptide stereochemistry. Prior efforts to discover DAACPs have been largely focused on pursuing peptides based on homology to known DAACPs or DAACP-encoding precursors. Here, we used a combination of enzymatic screening, mass spectrometry, and chromatographic analysis to identify novel DAACPs in the central nervous system (CNS) of Aplysia californica. We identified five new DAACPs from the pleurin precursor and three DAACPs from previously uncharacterized proteins. In addition, two peptides from the pleurin precursor, Plrn2 and Plrn3, exist as DAACPs with the d-residue found at position 2 or 3. These differentially modified forms of Plrn2 and Plrn3 are located in specific regions of the animal's CNS. Plrn2 and Plrn3 appear to be the first animal DAACPs in which the d-residue is found at more than one position, and this suggests that l- to d-residue isomerization may be a more variable/dynamic modification than previously thought. Overall, this study demonstrates the utility of nontargeted DAACP discovery approaches for identifying new DAACPs and demonstrates that isomerization is prevalent throughout the CNS of A. californica.