Cysteine-Based Coupling: Challenges and Solutions.

Antibody-drug conjugates (ADCs) have attracted great attention in recent years in the wake of an accelerated FDA approval rate and several large-scale acquisitions. To date, there are ten ADC drugs on the market and more than 70 in various stages of clinical trials. Yet, due to the complicated nature of ADC molecules, considerations need to cover many aspects for the success of ADCs, including target specificity, linker-payload stability, tumor permeability, and clearance rate. This topical review summarizes and discusses current methods used to increase stability and homogeneity of ADCs of cysteine conjugation. We believe that they will lead to improvement of efficacy and pharmacokinetics (PK) of ADC drugs.

The Role of SPECT and PET in Epilepsy.

OBJECTIVE. The purpose of this article is to summarize the role of molecular imaging of the brain by use of SPECT, FDG PET, and non-FDG PET radiotracers in epilepsy. CONCLUSION. Quantitative image analysis with PET and SPECT has increased the diagnostic utility of these modalities in localizing epileptogenic onset zones. A multi-modal platform approach integrating the functional imaging of PET and SPECT with the morphologic information from MRI in presurgical evaluation of epilepsy can greatly improve outcomes.

A hepatoprotection study of Radix Bupleuri on acetaminophen-induced liver injury based on CYP450 inhibition.

We investigated the potential hepatoprotective effect of Radix Bupleuri (RB) by inducing acute liver injury (ALI) in an animal model using acetaminophen (APAP) after pretreatment with RB aqueous extract for three consecutive days. Compared to those of the APAP group, the biochemical and histological results of the RB pretreatment group showed lower serumaspartate transaminase (AST) and alanine transaminase (ALT) levels as well as less liver damage. Pharmacokinetic study of the toxicity related marker acetaminophen-cysteine (APC) revealed a lower exposure level in rats, suggesting that RB alleviated APAP-induced liver damage by preventing glutathione (GSH) depletion. The results of cocktail approach showed significant inhibition of CYP2E1 and CYP3A activity. Further investigation revealed the increasing of CYP2E1 and CYP3A protein was significantly inhibited in pretreatment group, while no obvious effect on gene expression was found. Therefore, this study clearly demonstrates that RB exhibited significant protective action against APAP-induced acute live injury via pretreatment, and which is partly through inhibiting the increase of activity and translation of cytochrome P450 enzymes, rather than gene transcription.

Effect of toxoplasmic infection on the biodistribution of a brain radiopharmaceutical.

Purpose: This study evaluated if Toxoplasma gondii infection and the drug-associated infection modifies the brain radiopharmaceutical Ethylene Cystine Diethylester Dihydrochloride (99mTc-ECD) biodistribution in mice.Materials and methods: A total of 18 mice were divided into 3 groups. Control group (C) received distilled water and 99mTc-ECD; Infected group (I) received T. gondii strain and 99mTc-ECD; Infected and Treated group (IT), in addition to infection, received association of Pyrimethamine and Sulfadiazine and 99mTc-ECD. The T. gondii strain used in this study was TgCkRrRN3. Forty minutes after administration of the 99mTc-ECD, all animals were euthanized, and blood and brain samples were isolated. The counting of the radioactivity percentage per gram of tissue (%ATI/g) was calculated, and statistical analysis was performed by t-test, with a level of significance of p < .05.Results: There was a significant increase in %ATI/g between groups C and I on brain (0.35 ± 0.02 and 0.45 ± 0.04; p = .041) and on blood (0.80 ± 0.09 and 1.14 ± 0.31; p = .049). A significant decrease in %ATI/g occurred between groups C and IT on blood (0.80 ± 0.09 and 0.54 ± 0.08; p = .001) and on brain (0.35 ± 0.02 and 0.22 ± 0.04; p = .049).Conclusions: Combined therapy of anti-Toxoplasma drugs in infected mice reduced the uptake of 99mTc-ECD, probably due to its binding to plasma proteins.

Preparation of 99mTc-Labeled Mannan-S-Cysteine and Effect of Molecular Size of Mannan on Its Biodistribution.

Macrophage mannose receptor (MMR/CD206) is a promising target for the detection and identification of sentinel lymph node (SLN). MMR-targeting probes have been developed using mannosylated dextran, however, impairment of efficient targeting of SLN was often caused because of retention of injection site in which macrophages and dendritic cells exist. In this study, we prepared new MMR-targeting probes from yeast mannan (85 kDa), and its bioditribution was investigated. In-vivo evaluation showed that 11.9% of injected dose of 99mTc-labeled mannan-S-cysteines (99mTc-MSCs) was accumulated in popliteal lymph node (the SLN in this model), however, significant level of radioactivity (approximately 80%) was remained in injection site. Interestingly, 99mTc-labeled low molecular weight mannan-S-cysteine mannan (99mTc-LSC) prepared from 50 and 25 kDa mannan showed a decreased specific accumulation of 99mTc-LSC in the popliteal lymph node, while the radioactivity at the injection site remained unchanged. These results suggest that the molecular size, or nature/shape of the sugar chain is important for the specific accumulation of 99mTc-MSC in popliteal lymph node.

Changes in tissue gadolinium biodistribution measured in an animal model exposed to four chelating agents.

PURPOSE: This study investigated the potential to reduce gadolinium levels in rodents after repetitive IV Gadodiamide administration using several chelating agents. MATERIALS AND METHODS: The following six groups of rats were studied. Group 1: Control; Group 2: Gadodiamide only; Group 3: Meso-2,3-Dimercaptosuccinic acid (DMSA) + Gadodiamide; Group 4: N-Acetyl-L-cysteine (NAC) + Gadodiamide; Group 5: Coriandrum sativum extract + Gadodiamide; and Group 6: Deferoxamine + Gadodiamide. Brain, kidney, and blood samples were evaluated via inductively coupled plasma mass spectrometry. The brain was also evaluated histologically. RESULTS: Kidney gadolinium levels in Groups 4 and 5 were approximately double that of Group 2 (p = 0.033 for each). There was almost no calcification in rat hippocampus for Group 4 rodents when compared with Groups 2, 3, 5 and 6. CONCLUSION: Our preliminary study shows that excretion to the kidney has a higher propensity in NAC and Coriandrum sativum groups. It may be possible to change the distribution of gadolinium by administrating several agents. NAC may lower Gadodiamide-induced mineralization in rat hippocampus.

Absorption of 1-Dicysteinethioacetal-5-Hydroxymethylfurfural in Rats and Its Effect on Oxidative Stress and Gut Microbiota.

The absorption of a 5-hydroxymethylfurfural (HMF)-cysteine adduct, 1-dicysteinethioacetal-5-hydroxymethylfurfural (DCH), and its effect on antioxidant activity and gut microbiota were investigated. Results indicated that DCH is more easily absorbed in rats than HMF. Serum DCH concentrations were 15-38-fold of HMF concentrations from 30 to 180 min after intragastrical administration at the level of 100 mg/kg of body weight, and 2.7-4.5% of absorbed DCH was converted to HMF. The malondialdehyde content in the plasma, heart, liver, and kidneys significantly increased after drug (100 mg/kg of bw) administration for 1 week, suggesting that HMF and DCH were oxidative-stress-inducing agents, instead of antioxidant agents, in rats. HMF and DCH also modulated gut microbiota. HMF promoted the growth of Lactobacillus, Tyzzerella, Enterobacter, and Streptococcus. DCH increased the ratio of Firmicutes/ Bacteroidetes and promoted the growth of Akkermansia, Shigella, and Escherichia while inhibiting the growth of Lactobacillus.

Allicin Bioavailability and Bioequivalence from Garlic Supplements and Garlic Foods.

Allicin is considered responsible for most of the pharmacological activity of crushed raw garlic cloves. However, when garlic supplements and garlic foods are consumed, allicin bioavailability or bioequivalence (ABB) has been unknown and in question because allicin formation from alliin and garlic alliinase usually occurs after consumption, under enzyme-inhibiting gastrointestinal conditions. The ABB from 13 garlic supplements and 9 garlic foods was determined by bioassay for 13 subjects by comparing the area under the 32-h concentration curve of breath allyl methyl sulfide (AMS), the main breath metabolite of allicin, to the area found after consuming a control (100% ABB) of known allicin content: homogenized raw garlic. For enteric tablets, ABB varied from 36⁻104%, but it was reduced to 22⁻57% when consumed with a high-protein meal, due to slower gastric emptying. Independent of meal type, non-enteric tablets gave high ABB (80⁻111%), while garlic powder capsules gave 26⁻109%. Kwai garlic powder tablets, which have been used in a large number of clinical trials, gave 80% ABB, validating it as representing raw garlic in those trials. ABB did not vary with alliinase activity, indicating that only a minimum level of activity is required. Enteric tablets (high-protein meal) disintegrated slower in women than men. The ABB of supplements was compared to that predicted in vitro by the dissolution test in the United States Pharmacopeia (USP); only partial agreement was found. Cooked or acidified garlic foods, which have no alliinase activity, gave higher ABB than expected: boiled (16%), roasted (30%), pickled (19%), and acid-minced (66%). Black garlic gave 5%. The mechanism for the higher than expected ABB for alliinase-inhibited garlic was explored; the results for an alliin-free/allicin-free extract indicate a partial role for the enhanced metabolism of γ-glutamyl S-allylcysteine and S-allylcysteine to AMS. In conclusion, these largely unexpected results (lower ABB for enteric tablets and higher ABB for all other products) provide guidelines for the qualities of garlic products to be used in future clinical trials and new standards for manufacturers of garlic powder supplements. They also give the consumer an awareness of how garlic foods might compare to the garlic powder supplements used to establish any allicin-related health benefit of garlic.

Diversity in clinical manifestations and imaging features of neurosyphilis: obstacles to the diagnosis and treatment (report of three cases).

OBJECTIVE: To explore the clinical manifestations and imaging features of neurosyphilis and to discuss the obstacles in the diagnosis and treatment of neurosyphilis. METHODS: We present this case study involving three cases of definite neurosyphilis, focusing on their clinical data. RESULTS: Case 1 is a patient with numb and weak left lower limb. Case 2 showed slow reaction and dementia behaviors including worse memory and the decrease of calculation and orientation ability in this patient. Case 3 is a peripheral incomplete left oculomotor nerve palsy patient. Magnetic resonance imaging findings of three patients are different. And single photon emission computed tomography showed the regional cerebral blood flow was all hypoperfused. There were some difficulties in diagnosing and treating the patients in these three cases. CONCLUSION: The clinical manifestations and imaging findings of neurosyphilis are diverse. Clinicians should pay attention to neurosyphilis. After clear diagnosis, patients would receive norm treatment in time.

The efficacy of adrenocorticotropic hormone in a girl with anti-N-methyl-D-aspartate receptor encephalitis.

BACKGROUND: Immunomodulatory therapy has shown some therapeutic benefits in patients with anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis. In this report, we describe the use of adrenocorticotropic hormone (ACTH) immunotherapy with good outcome in a patient with anti-NMDAR encephalitis. SUBJECT AND METHODS: A 4-year-old girl developed convulsions in her right arm and leg without impaired consciousness. These convulsions occurred frequently in clusters of 10-20 events of 10-20 s duration. She was admitted to our hospital on the 6th day following her initial series of convulsions. Flaccid paralysis of the right hand and leg was also found. Interictal electroencephalography showed high-amplitude slow waves. No abnormal findings were shown on MRI. 99mTc-ECD brain SPECT on the 14th day showed hyperperfusion in the left hemisphere, including the left basal ganglia. The convulsions ceased following the oral administration of valproic acid on the 10th day; however, paralysis associated with choreic dyskinesia of the right arm and leg remained. ACTH immunotherapy was then performed on the 15th day. We identified the presence of N-methyl-D-aspartate receptor antibody in CSF samples taken on the 6th day. After ACTH therapy, the patient fully recovered from the paralysis associated with choreic dyskinesia of the right arm and leg. She has not had a relapse and has not required medication for over a year. CONCLUSION: ACTH immunotherapy may be a useful treatment option for patients with anti-NMDAR encephalitis, although further evaluation is required.

PET Imaging with S-[11C]Methyl-L-Cysteine and L-[Methyl-11C]Methionine in Rat Models of Glioma, Glioma Radiotherapy, and Neuroinflammation.

PURPOSE: S-[11C]-methyl-L-cysteine ([11C]MCYS) has been claimed to offer higher tumor selectivity than L-[methyl- 11C]methionine ([11C]MET). We examined this claim in animal models. PROCEDURES: Rats with implanted untreated (n = 10) or irradiated (n = 7, 1 × 25 Gy, on day 8) orthotopic gliomas were scanned after 6, 9, and 12 days, using positron emission tomography. Rats with striatal injections of saline (n = 9) or bacterial lipopolysaccharide (n = 9) were scanned after 3 days. RESULTS: Uptake of the two tracers in untreated gliomas was similar. [11C]MCYS was not accumulated in salivary glands, nasal epithelium, and healing wounds, in contrast to [11C]MET, but showed 40 % higher accumulation in the healthy brain. Both tracers showed a reduced tumor uptake 4 days after irradiation and minor accumulation in inflamed striatum. [11C]MCYS indicated higher lesion volumes than [11C]MET (untreated tumor + 47 %; irradiated tumor up to + 500 %; LPS-inflamed striatum + 240 %). CONCLUSIONS: [11C]MCYS was less accumulated in some non-tumor tissues than [11C]MET, but showed lower tumor-to-brain contrast.

Formulation of lyophilized single vial kit of N-N-Ethylene-L-Dicysteine (EC) for labeling with 99mTc. II: Radiochemical and clinical evaluation as a renal tubular agent in comparison with 99mTc-MAG3.

A Technetium 99mTc labeled lyophilized single component kit of N-N-ethylene-I-dicysteine (EC) is developed to replace multiple step kit developed by others. The aim of study is to formulate a radionuclide that is easy to prepare, has rapid plasma clearance, produce high quality images and is an affective alternative to radioiodine labeled orthoiodohippurate, which has been remained the physiological 'gold standard' since long time. To achieve this goal, the systematically varied key parameters such as pH, the use of reducing agents, stabilizers and additives are optimized to obtain maximum radiochemical purity and optimum biodistribution in non human and human primates. Various pH levels of EC showed equally good results in animal experiments but only pH 10 was suitable for human use. Dynamic and renal Scintigraphic studies are carried out with 99mTc-EC at pH 8 in 12 volunteers and at pH 10 in 18 volunteers and compared with 99mTc-MAG3, Background ratios, renograms, relative renal function and semi quantitative parameters are available in all studies. The background ratios (mean ± SD) at 30th minute are 0229±0.024 and 0.236±0.018 for 99mTc-EC at pH 10 and 99mTc-MAG3 respectively. The mean ± standard error of mean (SEM) values of TMAX and time to half activity (T12) for 99mTc-EC (pH10) are 3.7±0.6 and 7.3±1.0 respectively while for 99mTc-MAG3, they are 4.0±0.8 and 7.9±1.4 with p values 0.001 and 0.049 respectively. The values of relative renal function (RRF) for 99mTc-EC and 99mTc-MAG3 are 50.8±3.11 and 51.2±3.4 respectively with p value of 0.822. The residual activity at 25th minute (A25 / A MAX) and renal uptake are 0. 209±12.67±2.80 for 99mTc-EC and 0.218±0.035 and 1053±2.98 for 99mTc-MAG3 (p=0.031 an 0.0003) respectively. The correlation coefficient (R2) for TMAX, T1/2, A25/AMAX and renal uptake are 0.96, 0.69, 0.93 and 0.85 respectively.

Nanofitin as a New Molecular-Imaging Agent for the Diagnosis of Epidermal Growth Factor Receptor Over-Expressing Tumors.

Epidermal growth-factor receptor (EGFR) is involved in cell growth and proliferation and is over-expressed in malignant tissues. Although anti-EGFR-based immunotherapy became a standard of care for patients with EGFR-positive tumors, this strategy of addressing cancer tumors by targeting EGFR with monoclonal antibodies is less-developed for patient diagnostic and monitoring. Indeed, antibodies exhibit a slow blood clearance, which is detrimental for positron emission tomography (PET) imaging. New molecular probes are proposed to overcome such limitations for patient monitoring, making use of low-molecular-weight protein scaffolds as alternatives to antibodies, such as Nanofitins with better pharmacokinetic profiles. Anti-EGFR Nanofitin B10 was reformatted by genetic engineering to exhibit a unique cysteine moiety at its C-terminus, which allows the development of a fast and site-specific radiolabeling procedure with 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM). The in vivo tumor targeting and imaging profile of the anti-EGFR Cys-B10 Nanofitin was investigated in a double-tumor xenograft model by static small-animal PET at 2 h after tail-vein injection of the radiolabeled Nanofitin 18F-FBEM-Cys-B10. The image showed that the EGFR-positive tumor (A431) is clearly delineated in comparison to the EGFR-negative tumor (H520) with a significant tumor-to-background contrast. 18F-FBEM-Cys-B10 demonstrated a significantly higher retention in A431 tumors than in H520 tumors at 2.5 h post-injection with a A431-to-H520 uptake ratio of 2.53 ± 0.18 and a tumor-to-blood ratio of 4.55 ± 0.63. This study provides the first report of Nanofitin scaffold used as a targeted PET radiotracer for in vivo imaging of EGFR-positive tumor, with the anti-EGFR B10 Nanofitin used as proof-of-concept. The fast generation of specific Nanofitins via a fully in vitro selection process, together with the excellent imaging features of the Nanofitin scaffold, could facilitate the development of valuable PET-based companion diagnostics.

Oral Administration of (S)-Allyl-l-Cysteine and Aged Garlic Extract to Rats: Determination of Metabolites and Their Pharmacokinetics.

(S)-Allyl-l-cysteine is the major bioactive compound in garlic. (S)-Allyl-l-cysteine is metabolized to (S)-allyl-l-cysteine sulfoxide, N-acetyl-(S)-allyl-l-cysteine, and N-acetyl-(S)-allyl-l-cysteine sulfoxide after oral administration. An accurate LC-MS/MS method was developed and validated for the simultaneous quantification of (S)-allyl-l-cysteine and its metabolites in rat plasma, and the feasibility of using it in pharmacokinetic studies was tested. The analytes were quantified by multiple reaction monitoring using an atmospheric pressure ionization mass spectrometer. Because significant quantitative interference was observed between (S)-allyl-l-cysteine and N-acetyl-(S)-allyl-l-cysteine as a result of the decomposition of N-acetyl-(S)-allyl-l-cysteine at the detector source, chromatographic separation was required to discriminate (S)-allyl-l-cysteine and its metabolites on a reversed-phase C18 analytical column with a gradient mobile phase consisting of 0.1% formic acid and acetonitrile. The calibration curves of (S)-allyl-l-cysteine, (S)-allyl-l-cysteine sulfoxide, N-acetyl-(S)-allyl-l-cysteine, and N-acetyl-(S)-allyl-l-cysteine sulfoxide were linear over each concentration range, and the lower limits of quantification were 0.1 µg/mL [(S)-allyl-l-cysteine and N-acetyl-(S)-allyl-l-cysteine] and 0.25 µg/mL [(S)-allyl-l-cysteine sulfoxide and N-acetyl-(S)-allyl-l-cysteine sulfoxide]. Acceptable intraday and inter-day precisions and accuracies were obtained at three concentration levels. The method satisfied the regulatory requirements for matrix effects, recovery, and stability. The validated LC-MS/MS method was successfully used to determine the concentration of (S)-allyl-l-cysteine and its metabolites in rat plasma samples after the administration of (S)-allyl-l-cysteine or aged garlic extract.

Chemical and Biological Properties of S-1-Propenyl-l-Cysteine in Aged Garlic Extract.

S-1-Propenyl-l-cysteine (S1PC) is a stereoisomer of S-1-Propenyl-l-cysteine (SAC), an important sulfur-containing amino acid that plays a role for the beneficial pharmacological effects of aged garlic extract (AGE). The existence of S1PC in garlic preparations has been known since the 1960's. However, there was no report regarding the biological and/or pharmacological activity of S1PC until 2016. Recently, we performed a series of studies to examine the chemical, biological, pharmacological and pharmacokinetic properties of S1PC, and obtained some interesting results. S1PC existed only in trace amounts in raw garlic, but its concentration increased almost up to the level similar of SAC through aging process of AGE. S1PC showed immunomodulatory effects in vitro and in vivo, and reduced blood pressure in a hypertensive animal model. A pharmacokinetic study revealed that S1PC was readily absorbed after oral administration in rats and dogs with bioavailability of 88-100%. Additionally, S1PC had little inhibitory influence on human cytochrome P450 activities, even at a concentration of 1 mM. Based on these findings, S1PC was suggested to be another important, pharmacologically active and safe component of AGE similar to SAC. In this review, we highlight some results from recent studies on S1PC and discuss the potential medicinal value of S1PC.

Oral exposure of pregnant rats to toxic doses of methylmercury alters fetal accumulation.

Methylmercury (CH3Hg+) is an environmental toxicant that may lead to significant pathologies in exposed individuals. The current study assessed the disposition and toxicological effects of 2.5 or 7.5mgkg-1 CH3Hg+, conjugated to cysteine (Cys; Cys-S-CH3Hg) and administered orally to pregnant and non-pregnant Wistar and TR- rats. Rats were euthanized on gestational day 20 and the content of mercury in each fetus, amniotic sac, and placenta was determined. The brain, liver, and kidneys were removed from each fetus for estimation of mercury content. From the dams, a sample of blood, kidneys, liver, and brain were removed at the time of euthanasia. The findings from this study indicate that pregnancy leads to significant changes in the handling of mercuric ions, particularly in the liver. Furthermore, there are significant differences in the handling of non-nephrotoxic and nephrotoxic doses of Cys-S-CH3Hg by maternal and fetal organs.

Relationship between the change of language symptoms and the change of regional cerebral blood flow in the recovery process of two children with acquired aphasia.

OBJECTIVE: This study aimed to investigate the relationship between the change of language symptoms and the change of regional cerebral blood flow (rCBF) in the recovery process of two children with acquired aphasia caused by infarctions from Moyamoya disease with an onset age of 8years. METHODS: We compared the results for the Standard Language Test of Aphasia (SLTA) with rCBF changes in 7 language regions in the left hemisphere and their homologous regions in the right hemisphere at 4 time points from 3weeks for up to 5years after the onset of aphasia, while controlling for the effect of age. RESULTS: In both cases, strong correlations were seen within a hemisphere between adjacent regions or regions that are connected by neuronal fibers, and between some language regions in the left hemisphere and their homologous regions in the right hemisphere. Conversely, there were differences between the two cases in the time course of rCBF changes during their recovery process. CONCLUSION: Consistent with previous studies, the current study suggested that both hemispheres were involved in the long-term recovery of language symptoms in children with acquired aphasia. We suggest that the differences between both cases during their recovery process might be influenced by the brain states before aphasia, by which hemisphere was affected, and by the timing of the surgical revascularization procedure. However, the changes were observed in the data obtained for rCBF with strong correlations with the changes in language performance, so it is possible that rCBF could be used as a biomarker for language symptom changes.

Thermolysis kinetics and thermal degradation compounds of alliin.

To investigate thermolysis kinetics and identify degradation compounds, alliin solutions were heated at 60, 80, and 89°C. The degradation compounds of alliin were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS) and ultra-pressure liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The results showed that the thermal degradation kinetic of alliin could be described by a first-order reaction and k=4.38×1017exp (-142494/RT), where k is the reaction rate constant, min-1; R is gas constant; T is the absolute temperature, K. Degraded compounds, including S-allyl-l-cysteine and ethers, such as allyl alanine disulfide, allyl alanine trisulfide, allyl alanine tetrasulfide, dialanine disulfide (cysteine), dialanine trisulfide and dialanine tetrasulfide, were identified by HPLC-MS, MS/MS and UPLC-HRMS. Allyl alanine tetrasulfide was identified for the first time in alliin. The results show that alliin is unstable and significant numbers of organosulfur compounds are generated under high temperature treatment.

Nitrosothiol-Trapping-Based Proteomic Analysis of S-Nitrosylation in Human Lung Carcinoma Cells.

Nitrosylation of cysteines residues (S-nitrosylation) mediates many of the cellular effects of nitric oxide in normal and diseased cells. Recent research indicates that S-nitrosylation of certain proteins could play a role in tumor progression and responsiveness to therapy. However, the protein targets of S-nitrosylation in cancer cells remain largely unidentified. In this study, we used our recently developed nitrosothiol trapping approach to explore the nitrosoproteome of human A549 lung carcinoma cells treated with S-nitrosocysteine or pro-inflammatory cytokines. Using this approach, we identified about 300 putative nitrosylation targets in S-nitrosocysteine-treated A549 cells and approximately 400 targets in cytokine-stimulated cells. Among the more than 500 proteins identified in the two screens, the majority represent novel targets of S-nitrosylation, as revealed by comparison with publicly available nitrosoproteomic data. By coupling the trapping procedure with differential thiol labeling, we identified nearly 300 potential nitrosylation sites in about 150 proteins. The proteomic results were validated for several proteins by an independent approach. Bioinformatic analysis highlighted important cellular pathways that are targeted by S-nitrosylation, notably, cell cycle and inflammatory signaling. Taken together, our results identify new molecular targets of nitric oxide in lung cancer cells and suggest that S-nitrosylation may regulate signaling pathways that are critically involved in lung cancer progression.

The effective connectivity of the seizure onset zone and ictal perfusion changes in amygdala kindled rhesus monkeys.

Epileptic seizures are network-level phenomena. Hence, epilepsy may be regarded as a circuit-level disorder that cannot be understood outside this context. Better insight into the effective connectivity of the seizure onset zone and the manner in which seizure activity spreads could lead to specifically-tailored therapies for epilepsy. We applied the electrical amygdala kindling model in two rhesus monkeys until these animals displayed consistent stage IV seizures. At this stage, we investigated the effective connectivity of the amygdala by means of electrical microstimulation during fMRI (EM-fMRI). In addition, we imaged changes in perfusion during a seizure using ictal SPECT perfusion imaging. The spatial overlap between the connectivity network and the ictal perfusion network was assessed both at the regional level, by calculating Dice coefficients using anatomically defined regions of interest, and at the voxel level. The kindled amygdala was extensively connected to bilateral cortical and subcortical structures, which in many cases were connected multisynaptically to the amygdala. At the regional level, the spatial extents of many of these fMRI activations and deactivations corresponded to the respective increases and decreases in perfusion imaged during a stage IV seizure. At the voxel level, however, some regions showed residual seizure-specific activity (not overlapping with the EM-fMRI activations) or fMRI-specific activation (not overlapping with the ictal SPECT activations), indicating that frequently, only a part of a region anatomically connected to the seizure onset zone participated in seizure propagation. Thus, EM-fMRI in the amygdala of electrically-kindled monkeys reveals widespread areas that are often connected multisynaptically to the seizure focus. Seizure activity appears to spread, to a large extent, via these connected areas.