Sympathetic Blocks as a Predictor for Response to Ketamine Infusion in Patients with Complex Regional Pain Syndrome: A Multicenter Study.

BACKGROUND: Ketamine infusions are frequently employed for refractory complex regional pain syndrome (CRPS), but there are limited data on factors associated with treatment response. Sympathetic blocks are also commonly employed in CRPS for diagnostic and therapeutic purposes and generally precede ketamine infusions. OBJECTIVES: We sought to determine whether demographic and clinical factors, and technical and psychophysical characteristics of sympathetic blocks are associated with response to ketamine infusion. METHODS: In this multi-center retrospective study, 71 patients who underwent sympathetic blocks followed by ketamine infusions at 4 hospitals were evaluated. Sympathetically maintained pain (SMP) was defined as ≥ 50% immediate pain relief after sympathetic block and a positive response to ketamine was defined as ≥ 30% pain relief lasting over 3 weeks. RESULTS: Factors associated with a positive response to ketamine in univariable analysis were the presence of SMP (61.0% success rate vs 26.7% in those with sympathetically independent pain; P = .009) and post-block temperature increase (5.66 ± 4.20 in ketamine responders vs 3.68 ± 3.85 in non-responders; P = .043). No psychiatric factor was associated with ketamine response. In multivariable analysis, SMP (OR 6.54 [95% CI 1.83, 23.44]) and obesity (OR 8.75 [95% 1.45, 52.73]) were associated with a positive ketamine infusion outcome. CONCLUSIONS: The response to sympathetic blocks may predict response to ketamine infusion in CRPS patients, with alleviation of the affective component of pain and predilection to a positive placebo effect being possible explanations.

Normal-phase chromatographic separation of pigmented wine tannin by nano-HPLC quadrupole time-of-flight tandem mass spectrometry and identification of candidate molecular features.

BACKGROUND: As a wine ages, altered sensory properties lead to changes in perceived quality and value. Concurrent modifications of anthocyanin and tannin occur forming pigmented tannin, softening astringency and retaining persistent color. Wine tannin extracts of 1990 and 2010 vintages of Oakville Station Cabernet Sauvignon have been analyzed using normal-phase chromatography with tandem quadrupole time-of-flight mass spectrometry (QToF) to investigate the compositional differences in their pigmented tannin fractions. RESULTS: The older wine demonstrates much greater structural diversity and a range of more polar compounds, while the younger wine contains fewer observed ion peaks. Several hundred molecular features are observable, and, as expected, there is progression to higher molecular weights after long aging. Between 7% and 16% of molecular features could be matched to a database of anticipated pigmented tannin compounds. Many signals had multiple possible isomeric identities, but fragmentation to resolve their identity was stymied by low sensitivity of the tandem mass spectrometric capability provided by QToF, so isomeric disambiguation is incomplete. CONCLUSIONS: The chromatography displayed a high degree of resolution in aged wines, separating many of the known pigment types, including aldehyde bridged compounds, pyranoanthocyanins and direct condensation products among others, as well as resolving a great number of unknown compounds. Expanding our understanding of red wine pigments will lead to better wines as winemakers will be able to associate quality with particular wine pigment profiles once we can distinguish the relevant patterns in those pigments. © 2021 Society of Chemical Industry.

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM).

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.

Mechanistic peptidomics: factors that dictate specificity in the formation of endogenous peptides in human milk.

An extensive mass spectrometry analysis of the human milk peptidome has revealed almost 700 endogenous peptides from 30 different proteins. Two in-house computational tools were created and used to visualize and interpret the data through both alignment of the peptide quasi-molecular ion intensities and estimation of the differential enzyme participation. These results reveal that the endogenous proteolytic activity in the mammary gland is remarkably specific and well conserved. Certain proteins-not necessarily the most abundant ones-are digested by the proteases present in milk, yielding endogenous peptides from selected regions. Our results strongly suggest that factors such as the presence of specific proteases, the position and concentration of cleavage sites, and, more important, the intrinsic disorder of segments of the protein drive this proteolytic specificity in the mammary gland. As a consequence of this selective hydrolysis, proteins that typically need to be cleaved at specific positions in order to exert their activity are properly digested, and bioactive peptides encoded in certain protein sequences are released. Proteins that must remain intact in order to maintain their activity in the mammary gland or in the neonatal gastrointestinal tract are unaffected by the hydrolytic environment present in milk. These results provide insight into the intrinsic structural mechanisms that facilitate the selectivity of the endogenous milk protease activity and might be useful to those studying the peptidomes of other biofluids.

Current peptidomics: applications, purification, identification, quantification, and functional analysis.

Peptidomics is an emerging field branching from proteomics that targets endogenously produced protein fragments. Endogenous peptides are often functional within the body-and can be both beneficial and detrimental. This review covers the use of peptidomics in understanding digestion, and identifying functional peptides and biomarkers. Various techniques for peptide and glycopeptide extraction, both at analytical and preparative scales, and available options for peptide detection with MS are discussed. Current algorithms for peptide sequence determination, and both analytical and computational techniques for quantification are compared. Techniques for statistical analysis, sequence mapping, enzyme prediction, and peptide function, and structure prediction are explored.

A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

Site-specific glycosylation of secretory immunoglobulin A from human colostrum.

Secretory immunoglobulin A (sIgA) is a major glycoprotein in milk and plays a key role in mediating immune protection of the gut mucosa. Although it is a highly glycosylated protein, its site-specific glycosylation and associated glycan micro-heterogeneity have still not been fully elucidated. In this study, the site-specific glycosylation of sIgA isolated from human colostrum (n = 3) was analyzed using a combination of LC-MS and LC-MS/MS and in-house software (Glycopeptide Finder). The majority of the glycans found are biantennary structures with one or more acidic Neu5Ac residues; however, a large fraction belonged to truncated complex structures with terminal GlcNAc. Multiple glycosites were identified with nearly 30 glycan compositions located at seven sites on the secretory component, six compositions at a single site on the J chain, and 16 compositions at five sites on the IgA heavy (H) chain. Site-specific heterogeneity and relative quantitation of each composition and the extent of occupation at each site were determined using nonspecific proteases. Additionally, 54 O-linked glycan compositions located at the IgA1 hinge region (HR) were identified by comparison against a theoretical O-glycopeptide library. This represents the most comprehensive report to date detailing the complexity of glycan micro-heterogeneity with relative quantitation of glycoforms for each glycosylation site on milk sIgA. This strategy further provides a general method for determining site-specific glycosylation in large protein complexes.

Differential N-Glycosylation Patterns in Lung Adenocarcinoma Tissue.

To decrease the mortality of lung cancer, better screening and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and nonmalignant tissue at the level of individual glycan structures using nLC-chip-TOF-MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR < 0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, whereas levels of non- or low-galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to nonmalignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting, and biomarkers for lung cancer.

Endogenous human milk peptide release is greater after preterm birth than term birth.

BACKGROUND: Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile. OBJECTIVE: This study examined differences in peptides present between milk from women delivering at term and women delivering prematurely. METHODS: Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d). RESULTS: Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides. CONCLUSION: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant's immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127.

Peptidomic analysis of healthy and subclinically mastitic bovine milk.

A variety of proteases release hundreds of endogenous peptide fragments from intact bovine milk proteins. Mass spectrometry-based peptidomics allows for high throughput sequence assignment of a large number of these peptides. Mastitis is known to result in increased protease activity in the mammary gland. Therefore, we hypothesized that subclinically mastitic milks would contain higher concentrations of released peptides. In this work, milks were sampled from three cows and, for each, one healthy and one subclinically mastitic teat were sampled for milk. Peptides were analyzed by nano-liquid chromatography quadrupole time of flight tandem mass spectrometry and identified with database searching. In total, 682 peptides were identified. The total number of released peptides increased 146% from healthy to subclinically mastitic milks (p < 0.05), and the total abundance of released peptides also increased significantly (p < 0.05). Bioinformatic analysis of enzyme cleavage revealed increases in activity of cathepsin D and elastase (p < 0.05) with subclinical mastitis.

Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies.

Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides.

Whey permeate is a co-product obtained when cheese whey is passed through an ultrafiltration membrane to concentrate whey proteins. Whey proteins are retained by the membrane, whereas the low-molecular weight compounds such as lactose, salts, oligosaccharides and peptides pass through the membrane yielding whey permeate. Research shows that bovine milk from healthy cows contains hundreds of naturally occurring peptides - many of which are homologous with known antimicrobial and immunomodulatory peptides - and nearly 50 oligosaccharide compositions (not including structural isomers). As these endogenous peptides and oligosaccharides have low-molecular weight and whey permeate is currently an under-utilized product stream of the dairy industry, we hypothesized that whey permeate may serve as an inexpensive source of naturally occurring functional peptides and oligosaccharides. Laboratory fractionation of endogenous peptides and oligosaccharides from bovine colostrum sweet whey was expanded to pilot-scale. The membrane fractionation methodology used was similar to the methods commonly used industrially to produce whey protein concentrate and whey permeate. Pilot-scale fractionation was compared to laboratory-scale fractionation with regard to the identified peptides and oligosaccharide compositions. Results were interpreted on the basis of whether industrial whey permeate could eventually serve as a source of functional peptides and oligosaccharides. The majority (96%) of peptide sequences and the majority (96%) of oligosaccharide compositions found in the laboratory-scale process were mirrored in the pilot-scale process. Moreover, the pilot-scale process recovered an additional 33 peptides and 1 oligosaccharide not identified from the laboratory-scale extraction. Both laboratory- and pilot-scale processes yielded peptides deriving primarily from the protein ß-casein. The similarity of the laboratory-and pilot-scale's resulting peptide and oligosaccharide profiles demonstrates that whey permeate can serve as an industrial-scale source of bovine milk peptides and oligosaccharides.

Electrical isolation of dislocations in Ge layers on Si(001) substrates through CMOS-compatible suspended structures.

Suspended crystalline Ge semiconductor structures are created on a Si(001) substrate by a combination of epitaxial growth and simple patterning from the front surface using anisotropic underetching. Geometric definition of the surface Ge layer gives access to a range of crystalline planes that have different etch resistance. The structures are aligned to avoid etch-resistive planes in making the suspended regions and to take advantage of these planes to retain the underlying Si to support the structures. The technique is demonstrated by forming suspended microwires, spiderwebs and van der Pauw cross structures. We finally report on the low-temperature electrical isolation of the undoped Ge layers. This novel isolation method increases the Ge resistivity to 280 Ω cm at 10 K, over two orders of magnitude above that of a bulk Ge on Si(001) layer, by removing material containing the underlying misfit dislocation network that otherwise provides the main source of electrical conduction.

Modeling of external self-excitation and force generation on magnetic nanoparticles inside vitreous cavity.

The purpose of this manuscript was to design a better method for recovery from rhegmatogenous retinal detachment (RRD) surgery. We attempted to achieve this by designing a helmet that can manipulate intraocular magnetic nanoparticles (MNPs) and create a magnetic tamponade, eliminating the need for postoperative head positioning. A simulated analysis was developed to predict the pattern of magnetic force applied to the magnetic nanoparticles by external magnetic field. No participants were involved in this study. Instead, magnetic flux and force data for three different helmet designs were collected using virtual simulation tools. A prototype helmet was then constructed and magnetic flux and force data were recorded and compared to virtual data. For both virtual and physical scenarios, magnitude and direction of the resulting forces were compared to determine which design created the controlled direction and strongest forces into the back of the eye. Of the three virtual designs, both designs containing a visor had greater force magnitude than magnet alone. Between both designs with visors, the visor with bends resulted in forces more directed at the back of the eye. The physical prototype helmet shared similar measurements to virtual simulation with minimal percent error (Average = 5.47%, Standard deviation = 0.03). Of the three designs, the visor with bends generated stronger forces directed at the back of the eye, which is most appropriate for creating a tamponade on the retina. We believe that this design has shown promising capability for manipulating intraocular MNPs for the purpose of creating a tamponade for RRD.

Omics Forecasting: Predictive Calculations Permit the Rapid Interpretation of High-Resolution Mass Spectral Data from Complex Mixtures.

For some complex mixtures, chromatographic techniques are insufficient to separate the large numbers of compounds present. In addition, these mixtures often contain compounds with similar or identical molecular masses and shared fragmentation transitions. Advancements in mass spectrometry have provided more and more detailed molecular profiles with significant increases in resolution. This has led to a capacity to distinguish a very large number of compounds in complex mixtures, providing overwhelming data sets. The approach of calculating molecular formulas from a mass list has become more and more problematic as the number of signals has increased exponentially, to the point that it has become impossible to manually interpret the thousands of mass signals. The current approach is to calculate a list of possible formulas that fall within a specific mass error of the observed signal. Then, one must look for possible structures that can be derived from each entry on the list of formulas. However, an alternative approach is to anticipate the possible structures of a particular set of compounds, such as red wine pigments, and then compare the ion signals against a predicted list. To that end, starting with known wine pigment types, we have generated a set of expected wine pigment variants based on known derivatives of condensed tannin oligomers, anthocyanins, and fermentation products. After the ability to distinguish compounds by mass spectrometry was accounted for, over 1 million results were generated consisting of known and anticipated wine pigments. A comparison with a small sample of wine phenolic fractions show a large number of matches, suggesting that this approach may be helpful.

Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants.

BACKGROUND: Individuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the 'secretor' gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2'-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance. RESULTS: On average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2'-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ. CONCLUSIONS: Infants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants.

Peptidomic profile of milk of Holstein cows at peak lactation.

Bovine milk is known to contain naturally occurring peptides, but relatively few of their sequences have been determined. Human milk contains hundreds of endogenous peptides, and the ensemble has been documented for antimicrobial actions. Naturally occurring peptides from bovine milk were sequenced and compared with human milk peptides. Bovine milk samples from six cows in second-stage peak lactation at 78-121 days postpartum revealed 159 peptides. Most peptides (73%) were found in all six cows sampled, demonstrating the similarity of the intramammary peptide degradation across these cows. One peptide sequence, ALPIIQKLEPQIA from bovine perilipin 2, was identical to another found in human milk. Most peptides derived from ß-casein, αs1-casein, and αs2-casein. No peptides derived from abundant bovine milk proteins such as lactoferrin, ß-lactoglobulin, and secretory immunoglobulin A. The enzymatic cleavage analysis revealed that milk proteins were degraded by plasmin, cathepsins B and D, and elastase in all samples.