Nucleolin Malonylation as a Nuclear-Cytosol Signal Exchange Mechanism to Drive Cell Proliferation in Hepatocarcinoma by Enhancing AKT Translation.

Cancer cells undergo metabolic reprogramming that is intricately linked to malignancy. Protein acylations are especially responsive to metabolic changes, influencing signal transduction pathways and fostering cell proliferation. However, as a novel type of acylations, the involvement of malonylation in cancer remains poorly understood. In this study, we observed a significant reduction in malonyl-CoA levels in hepatocellular carcinoma (HCC), which correlated with a global decrease in malonylation. Subsequent nuclear malonylome analysis unveiled nucleolin (NCL) malonylation, which was notably enhanced in HCC biopsies. we demonstrated that NCL undergoes malonylation at lysine residues 124 and 398. This modification triggers the translocation of NCL from the nucleolus to nucleoplasm and cytoplasm, binding to AKT mRNA, and promoting AKT translation in HCC. Silencing AKT expression markedly attenuated HCC cell proliferation driven by NCL malonylation. These findings collectively highlight nuclear signaling in modulating AKT expression, suggesting NCL malonylation as a novel mechanism through which cancer cells drive cell proliferation.

Kinetics of Trisulfide-to-Disulfide Conversion of Therapeutic IgG1 Monoclonal Antibodies Under Physiological Conditions: A Case Study of Casirivimab And Imdevimab.

The percentage of trisulfide variants is a product quality metric that is monitored during the manufacture of monoclonal antibody (mAb)-based therapeutics. Results from earlier preclinical studies revealed that trisulfide linkages in mAbs are rapidly converted to disulfides in circulation. In this study, casirivimab and imdevimab, which are both IgG1 subclass mAbs that target the non-overlapping epitopes in SARS-CoV2 Spike protein, are used as models to study the kinetics of trisulfide-to-disulfide conversion in vivo in human circulation. To determine the percentage of trisulfide variants in systemic circulation immediately after intravenous injection, both mAbs were immunoprecipitated from serum samples collected from COVID-19 patients that received this cocktail antibody treatment as part of a first-in-human study. The immunoprecipitated mAbs were then digested under non-reducing conditions and evaluated by liquid-chromatography-mass spectrometry (LC-MS). Significant reductions in the percentages of trisulfide variants were observed in serum samples as early as 1 hr after completion of the intravenous infusion. A flow-through dialysis model designed to mimic the redox potential of blood revealed a plausible chemical mechanism for the rapid trisulfide-to-disulfide conversion of IgG1 subclass mAbs under physiological conditions.

Malonate differentially affects cell survival and confers chemoresistance in cancer cells via the induction of p53-dependent autophagy.

Metabolic network intertwines with cancerous signaling and drug responses. Malonate is a prevailing metabolite in cancer and a competitive inhibitor of succinate dehydrogenase (SDH). Recent studies showed that malonate induced reactive oxygen species (ROS)-dependent apoptosis in neuroblastoma cells, but protected cells from ischemia-reperfusion injury. We here revealed that malonate differentially regulated cell death and survival in cancer cells. While high-dose malonate triggered ROS-dependent apoptosis, the low-dose malonate induced autophagy and conferred resistance to multiple chemotherapeutic agents. Mechanistically, our results showed that malonate increased p53 stability and transcriptionally up-regulated autophagy modulator DRAM (damage-regulated autophagy modulator), thus promoting autophagy. We further proved that autophagy is required for malonate-associated chemoresistance. Collectively, our findings suggest that malonate plays a double-edge function in cancer response to stressors, and highlights a pro-cancer impact of p53-induced autophagy in response to malonate.

Identification of a small-molecule RPL11 mimetic that inhibits tumor growth by targeting MDM2-p53 pathway.

BACKGROUND: Targeting ribosome biogenesis to activate p53 has recently emerged as a therapeutic strategy in human cancer. Among various ribosomal proteins, RPL11 centralizes the nucleolar stress-sensing pathway by binding MDM2, leading to MDM2 inactivation and p53 activation. Therefore, the identification of MDM2-binding RPL11-mimetics would be valuable for anti-cancer therapeutics. METHODS: Based on the crystal structure of the interface between RPL11 and MDM2, we have identified 15 potential allosteric modulators of MDM2 through the virtual screening. RESULTS: One of these compounds, named S9, directly binds MDM2 and competitively inhibits the interaction between RPL11 and MDM2, leading to p53 stabilization and activation. Moreover, S9 inhibits cancer cell proliferation in vitro and in vivo. Mechanistic study reveals that MDM2 is required for S9-induced G2 cell cycle arrest and apoptosis, whereas p53 contributes to S9-induced apoptosis. CONCLUSIONS: Putting together, S9 may serve as a lead compound for the development of an anticancer drug that specifically targets RPL11-MDM2-p53 pathway.

Temporal and spatial dynamic propagation of electroencephalogram by combining power spectral and synchronization in childhood absence epilepsy.

Objective: During the transition from normal to seizure and then to termination, electroencephalography (EEG) signals have complex changes in time-frequency-spatial characteristics. The quantitative analysis of EEG characteristics and the exploration of their dynamic propagation in this paper would help to provide new biomarkers for distinguishing between pre-ictal and inter-ictal states and to better understand the seizure mechanisms. Methods: Thirty-three children with absence epilepsy were investigated with EEG signals. Power spectral and synchronization were combined to provide the time-frequency-spatial characteristics of EEG and analyze the spatial distribution and propagation of EEG in the brain with topographic maps. To understand the mechanism of spatial-temporal evolution, we compared inter-ictal, pre-ictal, and ictal states in EEG power spectral and synchronization network and its rhythms in each frequency band. Results: Power, frequency, and spatial synchronization are all enhanced during the absence seizures to jointly dominate the epilepsy process. We confirmed that a rapid diffusion at the onset accompanied by the frontal region predominance exists. The EEG power rapidly bursts in 2-4 Hz through the whole brain within a few seconds after the onset. This spatiotemporal evolution is associated with spatial diffusion and brain regions interaction, with a similar pattern, increasing first and then decreasing, in both the diffusion of the EEG power and the connectivity of the brain network during the childhood absence epilepsy (CAE) seizures. Compared with the inter-ictal group, we observed increases in power of delta and theta rhythms in the pre-ictal group (P < 0.05). Meanwhile, the synchronization of delta rhythm decreased while that of alpha rhythm enhanced. Conclusion: The initiation and propagation of CAE seizures are related to the abnormal discharge diffusion and the synchronization network. During the seizures, brain activity is completely changed with the main component delta rhythm. Furthermore, this article demonstrated for the first time that alpha inhibition, which is consistent with the brain's feedback regulation mechanism, is caused by the enhancement of the network connection. Temporal and spatial evolution of EEG is of great significance for the transmission mechanism, clinical diagnosis and automatic detection of absence epilepsy seizures.

Effectiveness and Safety of Oxcarbazepine vs. Levetiracetam as Monotherapy for Infantile Focal Epilepsy: A Longitudinal Cohort Study.

Objective: This study aimed to compare the effectiveness and safety of oxcarbazepine (OXC) vs. levetiracetam (LEV) for treating infantile focal epilepsy in a longitudinal cohort study. Methods: We enrolled 187 consecutive patients aged 2-24 months who received OXC or LEV as initial monotherapy; 161 patients completed the study. The longitudinal analysis involved anti-seizure medication (ASM) responsiveness, safety, the establishment of epilepsy syndrome, and etiology over a median follow-up of 2 years (interquartile range [IQR] 1.6-2.4). The relative efficacy and retention rates of OXC vs. LEV were evaluated using generalized linear regression models and the Cox proportional hazards model. Results: The 161 patients who completed the study had comparable baseline demographics and clinical variables between the OXC group (n = 83) and LEV group (n = 78). Overall, the mean age at onset was 6 months (IQR 4.3-9). The most common epilepsy syndrome was self-limited familial/non-familial infantile epilepsy (54.7%). Epilepsy was related to genetic and unknown causes in 34.2 and 52.2% of the patients, respectively. OXC achieved significantly higher responses than LEV for seizure freedom (risk ratio [RR] = 1.71, 95% confidence interval [CI] = 1.28-2.73, P < 0.001) and 12-month retention rate after onset (hazard ratio [HR] = 1.84, 95% CI = 1.15-2.95, P = 0.007). Moreover, OXC showed more obvious effects for patients aged < 1 year diagnosed with self-limited familial/non-familial infantile epilepsy and non-syndromic epilepsy with genetic or unknown causes. The adverse events related to both OXC and LEV were well-tolerated. Significance: OXC could be an alternative to LEV for treating infantile focal epilepsy. OXC monotherapy can be considered first-line treatment for patients aged <12 months and those with epilepsy without developmental and epileptic encephalopathy.

CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.

REGEN-COV® antibody cocktail bioanalytical strategy: comparison of LC-MRM-MS and immunoassay methods for drug quantification.

Aim: In response to the COVID-19 pandemic, Regeneron developed the anti-SARS-CoV-2 monoclonal antibody cocktail, REGEN-COV® (RONAPREVE® outside the USA). Drug concentration data was important for determination of dose, so a two-part bioanalytical strategy was implemented to ensure the therapy was rapidly available for use. Results & methodology: Initially, a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay, was used to analyze early-phase study samples. Subsequently, a validated electrochemiluminescence (ECL) immunoassay was implemented for high throughput sample analysis for all samples. A comparison of drug concentration data from the methods was performed which identified strong linear correlations and for Bland-Altman, small bias. In addition, pharmacokinetic data from both methods produced similar profiles and parameters. Discussion & conclusion: This novel bioanalytical strategy successfully supported swift development of a critical targeted therapy during the COVID-19 public health emergency.

Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Assay for Quantitative Measurement of Therapeutic Antibody Cocktail REGEN-COV Concentrations in COVID-19 Patient Serum.

REGEN-COV is a cocktail of two human IgG1 monoclonal antibodies (REGN10933 + REGN10987) that targets severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and has shown great promise to reduce the SARS-CoV-2 viral load in COVID-19 patients enrolled in clinical studies. A liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS)-based method, combined with trypsin and rAspN dual enzymatic digestion, was developed for the determination of total REGN10933 and total REGN10987 concentrations in several hundreds of pharmacokinetic (PK) serum samples from COVID-19 patients participating in phase I, II, and III clinical studies. The performance characteristics of this bioanalytical assay were evaluated with respect to linearity, accuracy, precision, selectivity, specificity, and analyte stability before and after enzymatic digestion. The developed LC-MRM-MS assay has a dynamic range from 10 to 2000 µg/mL antibody drug in the human serum matrix, which was able to cover the serum drug concentration from day 0 to day 28 after drug administration in two-dose groups for the clinical PK study of REGEN-COV. The concentrations of REGEN-COV in the two-dose groups measured by the LC-MRM-MS assay were comparable to the concentrations measured by a fully validated electrochemiluminescence (ECL) immunoassay.

Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation.

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

In Vivo Cross-Linking MS Reveals Conservation in OmpA Linkage to Different Classes of ß-Lactamase Enzymes.

Molecular interactions between two different classes of ß-lactamase enzymes and outer membrane protein A (OmpA) were studied by in vivo chemical cross-linking of a multi-drug-resistant strain of Acinetobacter baumannii AB5075. Class A ß-lactamase blaGES-11 and Class D ß-lactamase Oxa23, responsible for hydrolysis of different types of ß-lactam antibiotics, were found to be cross-linked to similar lysine sites of the periplasmic domain of outer membrane protein OmpA, despite low sequence homology between the two enzymes. The findings from in vivo XL-MS suggest that the interacting surfaces between both ß-lactamase enzymes and OmpA are conserved during molecular evolution, and the OmpA C-terminus domain serves an important function of anchoring different types of ß-lactamase enzymes in the periplasmic space.

Prednisolone/prednisone as adrenocorticotropic hormone alternative for infantile spasms: a meta-analysis of randomized controlled trials.

AIM: To compare the efficacy and safety of prednisolone/prednisone and adrenocorticotropic hormone (ACTH) in the treatment of infantile spasms using a meta-analysis of randomized controlled trials (RCTs). METHOD: In a systematic literature search of electronic databases (MEDLINE, Embase, the Cochrane Library), we identified RCTs that assessed prednisolone/prednisone compared with ACTH/tetracosactide in patients with infantile spasms. The electroclinical response and adverse events were evaluated. RESULTS: Six RCTs (616 participants) were included in the meta-analysis. Compared with prednisolone/prednisone, ACTH/tetracosactide was not superior in terms of cessation of spasms at day 14 (relative risk 1.19, 95% confidence interval [CI] 0.74-1.92), day 42 (relative risk 1.02, 95% CI 0.63-1.65), and resolution of hypsarrhythmia on electroencephalogram (relative risk 1.14, 95% CI 0.71-1.81); the incidences of common adverse reactions caused by ACTH/tetracosactide were not lower than that of prednisolone/prednisone for irritability (relative risk 0.79, 95% CI 0.57-1.10), increased appetite (relative risk 0.78, 95% CI 0.57-1.08), weight gain (relative risk 0.86, 95% CI 0.56-1.32), and gastrointestinal upset (relative risk 0.60, 95% CI 0.35-1.02), though it seemed less frequent. INTERPRETATION: Prednisolone/prednisone elicits a similar electroclinical response as ACTH for infantile spasms, which indicates that it can be an alternative to ACTH for treating infantile spasms. What this paper adds Prednisolone/prednisone is as effective as adrenocorticotropic hormone (ACTH) in electroclinical response of infantile spasms. Prednisolone/prednisone and ACTH cause similar and tolerable adverse effects, whose incidences are comparable. High-dose prednisone/prednisolone might be preferable to low dose for achieving freedom from spasms.

Prednisolona/prednisona como alternativa a la hormona adrenocorticotrópica para los espasmos infantiles: un metanálisis de ensayos controlados aleatorios OBJETIVO: Comparar la eficacia y seguridad de la prednisolona/prednisona y la hormona adrenocorticotrópica (ACTH) en el tratamiento de los espasmos infantiles mediante un metanálisis de ensayos controlados aleatorios (ECA). MÉTODO: En una búsqueda sistemática en la literatura de bases de datos electrónicas (MEDLINE, Embase, Cochrane Library), identificamos ECA que evaluaban prednisolona/ prednisona en comparación con ACTH/tetracosactida en pacientes con espasmos infantiles. Se evaluaron la respuesta electro clínica y los eventos adversos. RESULTADOS: Seis ECA (616 participantes) se incluyeron en el metanálisis. En comparación con la prednisolona/prednisona, la ACTH/tetracosactida no fue superior en términos de cese de espasmos en el día 14 (riesgo relativo 1,19, intervalo de confianza del 95% [IC] 0,74-1,92), día 42 (riesgo relativo 1,02, IC del 95% 0,63- 1,65), y la resolución de la hipsarritmia en el EEG (riesgo relativo 1,14, IC 95% 0,71-1,81); la incidencia de reacciones adversas comunes causadas por ACTH/tetracosactida no fue inferior a la de prednisolona/prednisona para irritabilidad (riesgo relativo 0,79, IC 95% 0,57-11,10), aumento del apetito (riesgo relativo 0,78, IC 95% 0,57-1,08), aumento de peso (riesgo relativo 0,86; IC del 95%: 0,56-1,32) y malestar gastrointestinal (riesgo relativo 0,60; IC del 95%: 0,35-1,02), aunque parecía menos frecuente. INTERPRETACIÓN: La prednisolona/prednisona provoca una respuesta electro clínica similar a la ACTH para los espasmos infantiles, lo que indica que puede ser una alternativa a la ACTH para el tratamiento de los espasmos infantiles.

Prednisolona/predinisona como hormônio adrenocorticotrópico alternativo para espasmos infantis: uma metanálise de estudos randomizados controlados OBJETIVO: Comparar a eficácia e segurança da prednisolona/ prednisona e hormônio adrenocorticotrópio (HACT) no tratamento de espasmos infantis usando uma metanálise de estudos randomizados controlados (ERCs). MÉTODO: Em uma busca sistemática da literatura em bases de dados eletrônicas (MEDLINE, Embase, Biblioteca Cochrane), identificamos ERCs que avaliaram a prednisolona/ prednisona em comparação com o HACT/ tetracosactídeo em pacientes com espasmos infantis. A resposta eletroclínica e eventos adversos foram avaliados. RESULTADOS: Seis ERCs (616 participantes) foram incluídos na metanálise. Comparado com a prednisolona/ prednisona , o HACT/ tetracosactídeo não foi superior em termos de cessação dos espasmos no dia 14 (risco relativo 1,19, intervalo de confiança [IC] a 95% 0,74-1,92), dia 42 (risco relativo 1,02, IC 95% 0,63-1,65), e resolução da hipsarritimia no EEG (risco relativo 1,14, IC 95% 0,71-1,81); as incidências de reações adversas comuns causadas pelo HACT/ tetracosactídeo não foram menores que as da prednisolona/ prednisona para irritabilidade (risco relativo 0,79, IC 95% 0,57-1010), aumento do apetite (risco relativo 0,78, IC 95% 0,57-1,08), ganho de peso (risco relativo 0,86, IC 95% 0,56-1,32), e mal-estar gastrointestinal (risco relativo 0,60, IC 95% 0,35-1,02), embora parecessem menos frequentes. INTERPRETAÇÃO: A prednisolona/ prednisona /prednisone elicia resposta eletroclínica similar ao HACT para espasmos infantis, o que indica que pode ser uma alternativa ao HACD para tratar espasmos infantis.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Novel SZT2 mutations in three patients with developmental and epileptic encephalopathies.

BACKGROUND: The seizure threshold 2 (SZT2) gene encodes a large, highly conserved protein that lowers seizure threshold and may also enhance epileptogenesis. In this study, three patients diagnosed with SZT2-related developmental and epileptic encephalopathies (DEEs) were reviewed aiming to expand knowledge of the genotype and phenotype of SZT2 mutations. METHODS: Targeted next-generation sequencing was performed to identify pathogenic mutations in 205 cases with DEEs of unknown etiology. Detailed clinical and genetic data were collected from SZT2-associated patients. RESULTS: Four novel mutations were found (c.1626 + 1G>A, c.5772dupA, c.4209C > A, c.7307_7308insG) in three patients. All the variants were inherited from their parents. Two patients were siblings and harbored the same mutations and presented developmental delay prior to the onset of seizures. All the individuals were diagnosed as DEEs, drug refractory epilepsy, and experienced status epilepticus (SE); one patient died of SE. One subject showed subependymal nodules as similar as those of tuberous sclerosis complex (TSC) in cranial magnetic resonance imaging (MRI). CONCLUSION: Our results expand the genotype and phenotypes of SZT2-related DEEs, suggesting that SZT2 mutations play a role in developmental delay and epileptic encephalopathy, with high susceptibility to SE and relatively specific MRI findings.

Systems structural biology measurements by in vivo cross-linking with mass spectrometry.

This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis.

Portable brain-computer interface based on novel convolutional neural network.

Electroencephalography (EEG) is a powerful, noninvasive tool that provides a high temporal resolution to directly reflect brain activities. Conventional electrodes require skin preparation and the use of conductive gels, while subjects must wear uncomfortable EEG hats. These procedures usually create a challenge for subjects. In the present study, we propose a portable EEG signal acquisition system. This study consists of two main parts: 1) A novel, portable dry-electrode and wireless brain-computer interface is designed. The EEG signal acquisition board is based on 24 bit, analog-to-digital converters chip and wireless microprocessor unit. The wireless portable brain computer interface device acquires an EEG signal comfortably, and the EEG signals are transmitted to a personal computer via Bluetooth. 2) A convolutional neural network (CNN) classification algorithm is implemented to classify the motor imagery (MI) experiment using novel feature 3-dimension input. The time dimension was reshaped to represent the first and second dimension, and the frequency band was used as the third dimension. Specifically, frequency domain representations of EEG signals obtained via wavelet package decomposition (WPD) are obtained to train CNN. The classification performance in terms of the value of kappa is 0.564 for the proposed method. The t-test results show that the performance improvement of CNN over other selected state-of-the-art methods is statistically significant. Our results show that the proposed design is reliable in measuring EEG signals, and the 3D CNN provides better classification performance than other method for MI experiments.

Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags: A Probe for Quantitative Glycomic Analysis.

Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.

Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry.

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.