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Background: CSF has long been accepted to circulate throughout the subarachnoid space, which lies between the arachnoid and pia maters of the meninges. How the CSF interacts with the cellular components of the developing postnatal meninges including the dura, arachnoid, and pia of both the meninges at the surface of the brain and the intracranial meninges, prior to its eventual efflux from the cranium and spine, is less understood. Here, we characterize small and large CSF solute distribution patterns along the intracranial and surface meninges in neonatal rodents and compare our findings to meningeal CSF solute distribution in a rodent model of intraventricular hemorrhage-posthemorrhagic hydrocephalus. We also examine CSF solute interactions with the tela choroidea and its pial invaginations into the choroid plexuses of the lateral, third, and fourth ventricles., Methods: 1.9-nm gold nanoparticles, 15-nm gold nanoparticles, or 3 kDa Red Dextran Tetramethylrhodamine constituted in aCSF were infused into the right lateral ventricle of P7 rats to track CSF circulation. 10 min post-1.9-nm gold nanoparticle and Red Dextran Tetramethylrhodamine injection and 4 h post-15-nm gold nanoparticle injection, animals were sacrificed and brains harvested for histologic analysis to identify CSF tracer localization in the cranial and spine meninges and choroid plexus. Spinal dura and leptomeninges (arachnoid and pia) wholemounts were also evaluated., Results: There was significantly less CSF tracer distribution in the dura compared to the arachnoid and pia maters in neonatal rodents. Both small and large CSF tracers were transported intracranially to the arachnoid and pia mater of the perimesencephalic cisterns and tela choroidea, but not the falx cerebri. CSF tracers followed a similar distribution pattern in the spinal meninges. In the choroid plexus, there was large CSF tracer distribution in the apical surface of epithelial cells, and small CSF tracer along the basolateral surface. There were no significant differences in tracer intensity in the intracranial meninges of control vs. intraventricular hemorrhage-posthemorrhagic hydrocephalus (PHH) rodents, indicating preserved meningeal transport in the setting of PHH., Conclusions: Differential CSF tracer handling by the meninges suggests that there are distinct roles for CSF handling between the arachnoid-pia and dura maters in the developing brain. Similarly, differences in apical vs. luminal choroid plexus CSF handling may provide insight into particle-size dependent CSF transport at the CSF-choroid plexus border., (© 2024. The Author(s).)

Posthemorrhagic hydrocephalus occurs in up to 30% of infants with high-grade intraventricular hemorrhage and is associated with the worst neurocognitive outcomes in preterm infants. The mechanisms of posthemorrhagic hydrocephalus after intraventricular hemorrhage are unknown; however, CSF levels of iron metabolic pathway proteins including hemoglobin have been implicated in its pathogenesis. Here, we develop an animal model of intraventricular hemorrhage using intraventricular injection of hemoglobin at post-natal day 4 that results in acute and chronic hydrocephalus, pathologic choroid plexus iron accumulation, and subsequent choroid plexus injury at post-natal days 5, 7, and 15. This model also results in increased expression of aquaporin-1, Na + /K + /Cl - cotransporter 1, and Na + /K + /ATPase on the apical surface of the choroid plexus 24 h post-intraventricular hemorrhage. We use this model to evaluate a clinically relevant treatment strategy for the prevention of neurological sequelae after intraventricular hemorrhage using intraventricular administration of the iron chelator deferoxamine at the time of hemorrhage. Deferoxamine treatment prevented posthemorrhagic hydrocephalus for up to 11 days after intraventricular hemorrhage and prevented the development of sensorimotor gating deficits. In addition, deferoxamine treatment facilitated acute iron clearance through the choroid plexus and subsequently reduced choroid plexus iron levels at 24 h with reversal of hemoglobin-induced aquaporin-1 upregulation on the apical surface of the choroid plexus. Intraventricular administration of deferoxamine at the time of intraventricular hemorrhage may be a clinically relevant treatment strategy for preventing posthemorrhagic hydrocephalus and likely acts through promoting iron clearance through the choroid plexus to prevent hemoglobin-induced injury., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus., (© 2023. The Author(s).)

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed. Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

Aim: Remote ischemic post conditioning (RIPOC) has shown to be neuroprotective against cerebral ischemic reperfusion (I/R) injury. However, the RIPOC protection against I/R injury induced cognitive abnormalities still remains elusive. Abundant evidence from earlier studies highlighted the role of heme oxygenase-1 (HO-1) in neuronal survival in various neurodegenerative disorders. Thus, in the present study, we investigated the possible contribution of HO-1 in RIPOC mediated neuroprotection against cerebral I/R injury and associated cognitive deficits., Experimental Procedure: Rats were subjected to bilateral common carotid occlusion model to induce I/R injury. RIPOC was achieved by 3 cycles of ischemia (10min) and reperfusion (10min) of bilateral femoral artery. Behavioral, biochemical and histological evaluation was performed. The levels of Tumor Necrosis Factor (TNF-α) were estimated. To further confirm molecular mechanism, HO-1 and Brain Derived Neurotrophic Factor (BDNF) activities were estimated., Results: Ischemic injury resulted in severe neurological deficits and cognitive abnormalities besides elevating oxidative stress and neuroinflammation. RIPOC intervention improved the behavioral parameters and anti-oxidant content. In addition, RIPOC decreased the levels of oxidative markers and pro-inflammatory cytokines like TNF-α. Moreover, RIPOC significantly upregulated HO-1 and neurotrophin including BDNF. Marked reduction in hippocampal structural abnormalities were observed with RIPOC intervention. SnPP treatment reversed the protective effects of RIPOC., Conclusion: These findings suggest that the neuroprotective effects of RIPOC during early reperfusion may be mediated through upregulation of HO-1 and BDNF, as the conditioning stimulus was found ineffective in presence of HO-1 inhibitor., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Remote limb ischemic post conditioning (RIPOC) has been reported to attenuate cerebral ischemic reperfusion (I/R) injury, while the molecular mechanisms remain elusive. Various studies have highlighted the involvement of glycogen synthase kinase (GSK-3β) in cerebral I/R injury and cognitive disorders. Hence, the present study was designed to explore the role of GSK-3β and its downstream regulators in RIPOC mediated neuroprotection against cerebral I/R injury and associated cognitive impairment. Male Wistar rats are randomly assigned into four groups: Sham, bilateral common carotid arteries occlusion (BCCAO), RIPOC and BCCAO+RIPOC. BCCAO was achieved by transient occlusion of bilateral common carotid arteries for 20min, followed by reperfusion. Non-invasive RIPOC was induced by 3 cycles each of 10min occlusion and reperfusion of both femoral arteries by using tourniquets, during early reperfusion phase. A battery of behavioral and cognitive tests were performed. Biochemical estimation of oxidative markers, anti-oxidants and pro-inflammatory markers were estimated. Levels of GSK-3β, CREB and BDNF were estimated to confirm the molecular mechanism. Hippocampal structural abnormalities were confirmed by H and E staining. The neurobehavioral analysis revealed that neurological and cognitive deficits caused by BCCAO, were reduced by RIPOC intervention. Meanwhile, the results of biochemical tests suggested that RIPOC attenuates the BCCAO induced oxidative damage, neuroinflammation and hippocampal structural abnormalities. Further, RIPOC prevented the elevation of BCCAO induced GSK-3β. RIPOC exerts neuroprotective effect against I/R injury, putatively by attenuating GSK-3β expression and upregulating the levels of CREB and BDNF., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aim: Numerous studies have demonstrated the possible neuroprotective role of lithium treatment against neurological disorders. However, the role of lithium in delayed phase of neuronal death against focal ischemia has not been explored. Therefore, the present study was designed to investigate the effect and molecular mechanisms of post-lithium treatment against cerebral ischemic reperfusion (I/R) injury and associated cognitive deficits in rats., Methods: I/R injury was induced by right middle cerebral artery occlusion and lithium (40 and 60 mg/kg) were given intraperitoneally, 24 h after the insult and continued for 1 week with 24-h interval. Using Lasser Doppler, cerebral blood flow was monitored before, during and after MCAO induction. Besides behavioral, biochemical, and histological evaluation, levels of tumor necrosis factor alpha (TNF-α) and brain-derived neurotrophic factor (BDNF) were also estimated., Results: I/R injury resulted in significant elevation of neurological deficits, oxidative stress, neuroinflammation, and cognitive impairments. We found that lithium injection, 24 h after I/R-injury continued for 1 week, dose dependently prevented behavioral abnormality and cognitive impairments. Moreover, lithium attenuated the levels of oxidative stress and pro-inflammatory-cytokines TNF-α level. Further, lithium treatments significantly reduced neuronal damage and augmented healthy neuronal count and improved neuronal density in hippocampus. These neuroprotective effects of delayed lithium treatment were associated with upregulation of neurotrophic factor BDNF levels., Conclusion: Delayed lithium treatment provides neuroprotection against cerebral I/R injury and associated cognitive deficits by upregulating BDNF expression that opens a new avenue to treat I/R injury even after active cell death.

Background: Hydroxy safflor yellow A (HSYA) has been translated clinically for cardiovascular diseases. HSYA is also greatly acknowledged for its protective effects against cerebral ischemic-reperfusion (I/R) injury. Although the precise mechanism of cerebral I/R injury is not fully understood, oxygen-derived free radicals and mitochondrial permeability transition pore (mPTP) opening during I/R injury are widely recognized as an important contributor to neuronal injury. Thus, we speculated that the neuroprotective effects of HSYA against cerebral I/R injury may be associated with mPTP modulation., Methods: Induction of I/R injury was achieved by 60 min of middle cerebral artery occlusion, followed by reperfusion for 24 h. For behavior and cognitive assessment, neurological scoring (NSS), rotarod, and Y-maze task were performed. Oxidative damage was measured in terms of markers such as malondialdehyde, reduced glutathione, and catalase levels and cerebral infarct volumes were quantified using 2,3,5-triphenyl tetrazolinium chloride staining. I/R injury-induced inflammation was determined using tumor necrosis factor-α (TNF-α) levels., Results: Animals exposed to I/R injury showed neurological severity, functional and cognitive disability, elevated oxidative markers, and TNF-α levels along with large infarct volumes. HSYA treatment during onset of reperfusion ameliorated performance in NSS, rotarod and Y-maze attenuated oxidative damage, TNF-α levels, and infarction rate. However, treatment with carboxyatractyloside, an mPTP opener, 20 min before HSYA, attenuated the protective effect of HSYA., Conclusions: Our study confirmed that protective effect of HSYA may be conferred through its free radical scavenger action followed by inhibiting the opening of mPTP during reperfusion and HSYA might act as a promising therapeutic agent against cerebral I/R injury.

Metabolic syndrome is increasingly recognized for its effects on cognitive health. Recent studies have highlighted the role of histone deacetylases (HDACs) in metabolic syndrome and cognitive functions. The present study was designed to investigate the possible therapeutic role of a HDAC inhibitor, trichostatin A (TSA), in cognitive impairment associated with metabolic syndrome. To ascertain the mechanisms involved, we fed mice with high-fat diet (HFD) for 4 weeks and examined changes in behavioral and biochemical/oxidative stress markers. Mice subjected to HFD exhibited characteristic features of metabolic disorder, viz., hyperglycemia, hypertriglyceridemia, hypercholesterolemia, and lower high-density lipoprotein (HDL) cholesterol levels. Moreover, these mice showed severe deficits in learning and memory as assessed by the Morris water maze and passive avoidance tasks along with elevated oxidative stress and inflammatory markers in brain homogenates. The observed changes occurred concurrently with reduced brain-derived neurotrophic factor (BDNF). In contrast, the mice treated with the HDAC inhibitor, TSA (0.5 and 1 mg/kg, i.p.), showed a significant and dose-dependent reduction in serum glucose, triglycerides, and total cholesterol along with improvement in HDL-cholesterol levels and learning and memory performance. TSA treatment also results in alleviation of oxidative stress and neuroinflammatory markers. Moreover, TSA significantly augmented the BDNF levels in HFD-fed mice. Thus, based upon these observations, it may be suggested that HDAC inhibition could be a novel therapeutic strategy to combat cognitive impairment associated with metabolic syndrome.

We report the preparation and characterization of monoolein cubosomes that can be easily surface modified through adsorption of a single layer of cationic poly-ε-lysine. Poly-ε-lysine coated cubosomes show remarkable stability in serum solution, are nontoxic and, are readily internalized by HeLa cells. The poly-ε-lysine coating provides chemical handles for further bioconjugation of the cubosome surface. We also demonstrate that the initial release rate of a hydrophilic drug, Naproxen sodium, from the cubosomes is retarded with just a single layer of polymer. Interestingly, cubosomes loaded with Naproxen sodium, recently shown to have anticancer properties, cause more apoptosis in HeLa cells when compared to free unencapsulated drug.

Background: Recent developments in LC-MS have turned it into a viable and valid alternative to ligand-binding assays. Large molecule bioanalysis by LC-MS is generally performed by tryptic digestion, purification and detection of one or more small signature peptides. High-resolution MS instruments offer quantification of intact small proteins or peptides and are able to increase the selectivity, while maintaining sensitivity., Results: Unlike multiple reaction monitoring assays, several factors affecting data processing were presented and the optimal parameters to consider during quantification method building were also demonstrated. MUC5AC-13 (MW 1709.8 Da), human hepcidin/LEAP-1 (MW 2797.4 Da), porcine calcitonin (MW 3604 Da) and chicken lysozyme (MW 14.3 kDa) were selected as model compounds and the possibility of intact peptide and small protein quantification, without tryptic digestion, was demonstrated., Conclusion: Selectivity and sensitivity were improved using different scan modes, such as TOF-MS and TOF-MS/MS.

It can be argued that the last true paradigm shift in the bioanalytical (BA) arena was the shift from high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection to HPLC with tandem mass spectrometry (MS/MS) detection after the commercialization of the triple quadrupole mass spectrometer in the 1990s. HPLC-MS/MS analysis based on selected reaction monitoring (SRM) has become the gold standard for BA assays and is used by all the major pharmaceutical companies for the quantitative analysis of new drug entities (NCEs) as part of the new drug discovery and development process. While LC-MS/MS continues to be the best tool for drug discovery bioanalysis, a new paradigm involving high-resolution mass spectrometry (HRMS) and ultrahigh-pressure liquid chromatography (uHPLC) is starting to make inroads into the pharmaceutical industry. The ability to collect full scan spectra, with excellent mass accuracy, mass resolution, 10-250 ms scan speeds and no NCE-related MS parameter optimization, makes the uHPLC-HRMS techniques suitable for quantitative analysis of NCEs while preserving maximum qualitative information about other drug-related and endogenous components such as metabolites, degradants, biomarkers and formulation materials. In this perspective article, we provide some insight into the evolution of the hybrid quadrupole-time-of-flight (Qq-TOF) mass spectrometer and propose some of the desirable specifications that such HRMS systems should have to be integrated into the drug discovery bioanalytical workflow for performing integrated qualitative and quantitative bioanalysis of drugs and related components., (Copyright © 2011 John Wiley & Sons, Ltd.)

Octadecenyl thiophosphate (OTP), a synthetic analogue of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD(80/30) radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid-liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1-->95.0 and 403.1-->95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Quantification of alpha- and gamma-endorphins in rat brain using liquid chromatography-electrospray ionization-tandem mass spectrometry is described. [D-Ala(2)]-gamma-endorphin is used as an internal standard. The precursor-to-product ion MRM transitions for alpha-endorphin, gamma-endorphin, and [D-Ala(2)]-gamma-endorphin were m/z 873.6-->429.6; 929.6-->542.3; 936.6-->542.3, respectively. The method was validated in terms of linearity, specificity, sensitivity, recovery, precision, and accuracy. The assay was linear over a concentration range of 0.1-200 ng/mL with the limit-of-detection of 0.03 ng/mL and limit-of-quantification of 0.1 ng/mL. The endogenous concentrations of alpha- and gamma-endorphins in rat brains were 13.8+/-0.57 (mean+/-SD; n=5) and 2.5+/-0.43 ng/g of wet tissue weight, respectively.

Our group has used the tetrahydroisoquinoline derivative EDL-155 to treat glioblastoma in animal models and it is currently being evaluated in the treatment of ocular cancers. The purpose of this study was to develop a rapid and sensitive liquid chromatography and tandem mass spectrometry (LC-MS/MS) method to study the plasma and vitreous humor disposition of EDL-155 in rats. Animals received a single periocular injection of EDL-155 (20 mg/kg). Animals were sacrificed at specified times (5, 60, 120, 240 and 360 min) and plasma and vitreous humor samples were obtained. EDL-155 was isolated by protein precipitation and the extracts were analyzed by reversed-phase high-pressure liquid chromatography (HPLC) with MS/MS detection. A structurally similar analog was used as internal standard (IS). The chromatographic run time was 3.5 min per injection. The mass spectrometer was operated in positive-ion, multiple reaction monitoring (MRM) mode. The mass transitions monitored were m/z332.2 --> 167.2 (EDL-155) and m/z391.2 --> 200.2 (IS). The lower limit of quantification (LLOQ) was 0.1 ng/ml in both vitreous humor and plasma. The method was validated for selectivity, linearity, accuracy and precision in rat vitreous humor and partially validated for accuracy and precision in rat plasma. The ion suppression, recovery and stability of the analyte in the biological matrix were also tested. The assay was rapid, sensitive and robust enough to support EDL-155 ocular penetration studies in a rodent model of intraocular cancer. Application of this method revealed that EDL-155 was rapidly passed into the vitreous humor following periocular administration. Further, vitreous humor exposure exceeded systemic exposure by approximately sevenfold. High local concentrations coupled with minimal systemic exposure supports further testing of EDL-155 as localized therapy for intraocular cancers., (2009 John Wiley & Sons, Ltd.)

Purpose: Tetrahydroisoquinolines (THIs) have demonstrated anti-cancer activity in rodent models of glioma, a form of brain cancer refractory to therapeutic intervention. In this study, peripheral and cerebrospinal fluid (CSF) pharmacokinetics in rats were determined to assess the drug developability of the novel THI EDL-155 for the treatment of glioma., Methods: Serial blood and CSF samples were collected from rats following intravenous bolus administration of EDL-155 (10-20 mg/kg). Samples were analyzed by LC/MS/MS. Pharmacokinetic analyses using compartmental and noncompartmental methods were performed using the computer program WinNonlin. Plasma protein binding was measured using the charcoal adsorption method. The in vivo efficacy of EDL-155 (i.p. 20 mg/kg twice daily for 7 days) was assessed in rats with stereotactically implanted C6 glioma cells into the caudate., Results: EDL-155 plasma concentration data were described by a one-compartment model. EDL-155 demonstrated rapid clearance (342.5+/-49.9 ml/min/kg), high volume of distribution (13.0+/-1.2 l/kg) and a terminal half-life of 23.7+/-1.5 min. Dose-normalized CSF area under the curve (AUC(CSF)) as a percentage of peripheral exposure (AUC(Plasma)) was 1.4%. EDL-155 was highly bound to plasma proteins (>93%). Intracranial tumor volume at 7 days post-implantation was approximately 30% smaller in animals treated with EDL-155 when compared to vehicle control animals (13.2+/-5.3 mm(3) vs. 18.7+/-6.3 mm(3); P=0.04)., Conclusion: High clearance and extensive protein binding limit the brain availability of EDL-155 following systemic administration. EDL-155 treatment resulted in reduced tumor size despite limited blood brain barrier penetrability, which suggests that analogs with increased metabolic stability and brain penetrability may provide a therapeutic option for primary central nervous system tumors such as glioma. On-going studies are focused on the design, synthesis, and testing of novel analogs based upon these findings.

A rapid method for quantitative chiral analysis of phthaloylglutamic acid and its dimethyl ester by Cook's kinetic method is demonstrated using electrospray ionization (ESI) and matrix-assisted laser desorption techniques. Transition-metal-bound complex ions containing the chiral phthaloylglutamic acid and its dimethyl ester are generated by ESI mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the drug mixture. A seven-point calibration curve, derived from the kinetic method, allowed rapid quantitation of the enantiomeric excess of drug mixtures. In this paper, matrix-assisted laser desorption/ionization (MALDI) coupled with the linear ion trap (LIT) technique is evaluated for its applicability as a complementary technique to ESI for chiral discrimination and quantitation.

Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.

In the present work, we report conversion of fluoxetine (Prozac), a novel anti depressant to N-methyl fluoxetine in formalin fixed liver tissue. Earlier studies indicate that drugs containing secondary amino group will react with formalin to form corresponding N-methyl derivatives. Even though embalming cadavers is common, it may create problems for forensic toxicologists if a case was not previously suspected. In formalin solutions, fluoxetine is methylated producing N-methyl fluoxetine. N-Methyl fluoxetine standard was synthesized by treating fluoxetine in formaldehyde solution. The structure confirmed by (1)HNMR and gas chromatography-mass spectrometry in electron impact ionization mode. Randomly chosen rat liver pieces (200-250 mg) were injected with 100 microg of Fluoxetine. The liver pieces were covered with three different concentrations of formalin, 5%, 10%, and 20%, and at three different pHs, 3.0, 7.0, and 9.5. The reaction was studied for a total period of 30 days, and the reaction products were monitored on days 0, 4, 14, and 30 days. The study indicates that the rate of conversion of fluoxetine to its N-methyl derivative increased with increase in the concentration of formalin and pH of the solution. The conversion is rapid at higher pH values. Fluoxetine was totally converted to its N-methyl derivatives after 30 days in 20% formalin at pH 9.5. Therefore, analysis for parent drug or its N-methyl derivative in embalmed tissues may provide data that will reduce the likelihood of false negatives.

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.

Dynamin is a GTPase protein that is essential for clathrin-mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi(ts1) changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton-X extraction. Addition of Na(+) or K(+) can extract dynamin to the cytosolic (supernatant) fraction. The shi(ts1) mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature-sensitive alleles or wild type. Sensitivity to salt extraction in shi(ts1) was enhanced by GTP and nonhydrolyzable GTP-gammaS. The shi(ts1) mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature-sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi(ts1) flies recovered much more slowly from heat-induced paralysis than did other temperature-sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd(msf15)), which reduces the paralytic temperature threshold of other temperature-sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi(ts1). Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses., (Copyright 2002 Wiley Periodicals, Inc.)