Peer-led intervention for individuals with major depression: study protocol for a randomized controlled trial (SUPEERMood).

BACKGROUND: Major Depressive Disorder (MDD) is one of the most disabling mental health problems worldwide. The Recovery Model emphasizes peer support to empower individuals with MDD, improve self-management, and patients' quality of life. Despite the demonstrated efficacy of peer-led interventions, further research is needed due to methodological limitations and variability in interventions across studies. Therefore, the objective of this trial is to evaluate the effectiveness of an adjuvant peer-led intervention for the reduction of depressive symptoms in individuals diagnosed with MDD attended in primary care mental health units. METHODS: A controlled, parallel, randomized clinical trial will be conducted. The intervention group (n = 35) will receive 6 weeks of peer-led sessions based on a peer support program drive whilst supervised by nurses, while the control group (n = 35) will use a mobile Health (mHealth) application for emotional wellness based on CBT for 6 weeks. Measurements will be collected at baseline, at 6 weeks, at 6 and 12 months after the intervention to evaluate post-intervention effects. The primary outcome is the reduction of depressive symptoms through the Beck Depression Inventory (BDI-II) after the intervention. Secondary outcomes will involve measures such as adherence to psychiatric treatment, quality of life, adherence to mediterranean diet, alcohol consumption and physical activity. DISCUSSION: We hypothesize that this peer-led intervention, in contrast to the mHealth, will show improvement in BDI-II score reduction of 6 points after six weeks, 6 and 12 months. Standardized peer-led programs can benefit patients and professionals in terms of efficacy and feasibility of clinical treatment of depression, healthy habits, self-care and quality of life. In addition, they can provide recovery and relapse reduction, improved psychosocial support, minimization of intensive care use, and support for patient autonomy through self-management. TRIAL REGISTRATION: The trial protocol is prospectively registered with ClinicalTrials.gov under protocol registration number NCT06398561. Date of registration: May 01, 2024. Recruitment is ongoing.

Protocol for a seamless phase 2A-phase 2B randomized double-blind placebo-controlled trial to evaluate the safety and efficacy of benfotiamine in patients with early Alzheimer's disease (BenfoTeam).

BACKGROUND: Benfotiamine provides an important novel therapeutic direction in Alzheimer's disease (AD) with possible additive or synergistic effects to amyloid targeting therapeutic approaches. OBJECTIVE: To conduct a seamless phase 2A-2B proof of concept trial investigating tolerability, safety, and efficacy of benfotiamine, a prodrug of thiamine, as a first-in-class small molecule oral treatment for early AD. METHODS: This is the protocol for a randomized, double-blind, placebo-controlled 72-week clinical trial of benfotiamine in 406 participants with early AD. Phase 2A determines the highest safe and well-tolerated dose of benfotiamine to be carried forward to phase 2B. During phase 2A, real-time monitoring of pre-defined safety stopping criteria in the first approximately 150 enrollees will help determine which dose (600 mg or 1200 mg) will be carried forward into phase 2B. The phase 2A primary analysis will test whether the rate of tolerability events (TEs) is unacceptably high in the high-dose arm compared to placebo. The primary safety endpoint in phase 2A is the rate of TEs compared between active and placebo arms, at each dose. The completion of phase 2A will seamlessly transition to phase 2B without pausing or stopping the trial. Phase 2B will assess efficacy and longer-term safety of benfotiamine in a larger group of participants through 72 weeks of treatment, at the selected dose. The co-primary efficacy endpoints in phase 2B are CDR-Sum of Boxes and ADAS-Cog13. Secondary endpoints include safety and tolerability measures; pharmacokinetic measures of thiamine and its esters, erythrocyte transketolase activity as blood markers of efficacy of drug delivery; ADCS-ADL-MCI; and MoCA. CONCLUSION: The BenfoTeam trial utilizes an innovative seamless phase 2A-2B design to achieve proof of concept. It includes an adaptive dose decision rule, thus optimizing exposure to the highest and best-tolerated dose. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT06223360, registered on January 25, 2024. https://classic.clinicaltrials.gov/ct2/show/NCT06223360.

Compassionate Care: A Qualitative Exploration of Nurses' Inner Resources in the Face of Burnout.

There is a universal shortage of nurses, with a current needs-based shortage of 5.9 million. This is not solely a recruitment issue but one of retention, triggered by high levels of work-induced stress, burnout, and reports of low job satisfaction resulting in poor care delivery. Some of the health repercussions on nurses include anxiety, insomnia, depression, migraines, irritability, absenteeism, and sometimes alcoholism and drug abuse. To tackle some of these costly issues, a qualitative exploration into how inner resources is used by nurses to cope with stress at different points of their careers is proposed. Through the lens of grounded theory, semi-structured interviews will be carried out with two distinct sets of participants: (1) Student nurses registered at the University of the Illes Baleares between 2022-2025. (2) Experienced nurses on the Balearic nursing register. Interviews will be coded and then analysed using Atlas.ti. Expected results will inform curriculum improvements that will benefit the well-being of (student) nurses, from the outset of their training, pre-empting potential psycho-social risks before they arise in the workplace. This is vital as it addresses nurses' mental health as well as chronic issues of retention and absenteeism.

Uncovering the dominant role of root metabolism in shaping rhizosphere metabolome under drought in tropical rainforest plants.

Plant-soil-microbe interactions are crucial for driving rhizosphere processes that contribute to metabolite turnover and nutrient cycling. With the increasing frequency and severity of water scarcity due to climate warming, understanding how plant-mediated processes, such as root exudation, influence soil organic matter turnover in the rhizosphere is essential. In this study, we used 16S rRNA gene amplicon sequencing, rhizosphere metabolomics, and position-specific 13C-pyruvate labeling to examine the effects of three different plant species (Piper auritum, Hibiscus rosa sinensis, and Clitoria fairchildiana) and their associated microbial communities on soil organic carbon turnover in the rhizosphere. Our findings indicate that in these tropical plants, the rhizosphere metabolome is primarily shaped by the response of roots to drought rather than direct shifts in the rhizosphere bacterial community composition. Specifically, the reduced exudation of plant roots had a notable effect on the metabolome of the rhizosphere of P. auritum, with less reliance on neighboring microbes. Contrary to P. auritum, H. rosa sinensis and C. fairchildiana experienced changes in their exudate composition during drought, causing alterations to the bacterial communities in the rhizosphere. This, in turn, had a collective impact on the rhizosphere's metabolome. Furthermore, the exclusion of phylogenetically distant microbes from the rhizosphere led to shifts in its metabolome. Additionally, C. fairchildiana appeared to be associated with only a subset of symbiotic bacteria under drought conditions. These results indicate that plant species-specific microbial interactions systematically change with the root metabolome. As roots respond to drought, their associated microbial communities adapt, potentially reinforcing the drought tolerance strategies of plant roots. These findings have significant implications for maintaining plant health and preference during drought stress and improving plant performance under climate change.

An experimental, computational, and uncertainty analysis study of the rates of iodoalkane trapping by DABCO in solution phase organic media.

NMR spectroscopy was used to measure the rates of the first and second substitution reactions between iodoalkane (R = Me, 1-butyl) and DABCO in methanol, acetonitrile and DMSO. Most of the reactions were recorded at three different temperatures, which permitted calculation of the activation parameters from Eyring and Arrhenius plots. Additionally, the reaction rate and heat of reaction for 1-iodobutane + DABCO in acetonitrile and DMSO were also measured using calorimetry. To help interpret experimental results, ab initio calculations were performed on the reactant, product, and transition state entities to understand structures, reaction enthalpies and activation parameters. Markov chain Monte Carlo statistical sampling was used to determine a distribution of kinetic rates with respect to the uncertainties in measured concentrations and correlations between parameters imposed by a kinetics model. The reactions with 1-iodobutane are found to be slower in all cases compared to reactions under similar conditions for iodomethane. This is due to steric crowding around the reaction centre for the larger butyl group compared to methyl which results in a larger activation energy for the reaction.

Pharmacological thiamine levels as a therapeutic approach in Alzheimer's disease.

of the study.

The Spanish Version of the State Self-Compassion Scale-Long Form (SSCS-L): A Study of Its Validity and Reliability in a Sample of Nursing Students.

BACKGROUND: In nurses, self-compassion mitigates the effects of stress, burnout and compassion fatigue, and enhances empathy, compassion and well-being and quality of life. The Self-Compassion Scale is the most-used instrument. The aim of this study is to validate the Spanish version of the new developed State Self-Compassion Scale-Long (SSCS-L). METHODS: Students of the first year of the Nursing Degree were surveyed online. Together with the SSCS-L, their levels of positive and negative affect was reported. Analyses included descriptive statistics, competitive confirmatory factor analysis, evidence on criterion-related validity and estimates of reliability. RESULTS: The best fitting model for the SSCS-L was the one hypothesizing six-correlated factors of self-compassion: self-kindness, common humanity, mindfulness, self-judgement, isolation, and over-identification. Positive relations between the positive dimensions of self-compassion and positive affect were found, whereas there were negative relations between the positive poles of self-compassion and negative affect. Estimates of reliability were adequate, except for the dimension of over-identification. CONCLUSIONS: Self-compassion has become a key competency for nurses. The SSCS-L is an appropriate tool to allow an adequate assessment of self-compassion in experimental contexts.

Elucidating Drought-Tolerance Mechanisms in Plant Roots through 1H NMR Metabolomics in Parallel with MALDI-MS, and NanoSIMS Imaging Techniques.

As direct mediators between plants and soil, roots play an important role in metabolic responses to environmental stresses such as drought, yet these responses are vastly uncharacterized on a plant-specific level, especially for co-occurring species. Here, we aim to examine the effects of drought on root metabolic profiles and carbon allocation pathways of three tropical rainforest species by combining cutting-edge metabolomic and imaging technologies in an in situ position-specific 13C-pyruvate root-labeling experiment. Further, washed (rhizosphere-depleted) and unwashed roots were examined to test the impact of microbial presence on root metabolic pathways. Drought had a species-specific impact on the metabolic profiles and spatial distribution in Piper sp. and Hibiscus rosa sinensis roots, signifying different defense mechanisms; Piper sp. enhanced root structural defense via recalcitrant compounds including lignin, while H. rosa sinensis enhanced biochemical defense via secretion of antioxidants and fatty acids. In contrast, Clitoria fairchildiana, a legume tree, was not influenced as much by drought but rather by rhizosphere presence where carbohydrate storage was enhanced, indicating a close association with symbiotic microbes. This study demonstrates how multiple techniques can be combined to identify how plants cope with drought through different drought-tolerance strategies and the consequences of such changes on below-ground organic matter composition.

Polychlorinated biphenyls induce oxidative stress and metabolic responses in astrocytes.

Exposure to environmental toxicants is prevalent, hazardous and linked to varied detrimental health outcomes and disease. Polychlorinated biphenyls (PCBs), a class of hazardous organic chlorines once widely used for industrial purposes, are associated with neurodegenerative disease and oxidative stress in both in vitro and in vivo models. Here, we investigated the impact of Aroclor 1254, a commercially available PCB mixture, on primary murine astrocytes to determine the response to this once ubiquitously used toxicant on the most numerous cells of the central nervous system (CNS). Astrocytes are a critical component of homeostasis throughout the CNS, including at the blood-brain barrier, where they serve as the primary defense against xenobiotics entering the CNS, and at the synapse, where they are closely coupled to neurons through several metabolic pathways. We hypothesized that PCBs cause astrocytic oxidative stress and related dysfunction including altered metabolism. We exposed primary murine cortical astrocytes to PCBs and report an increased expression of antioxidant genes (Prdx1, Gsta2, Gfap, Amigo2) in response to oxidative stress. Our data show increased ATP production and spare respiratory capacity in astrocytes exposed to 10 µM (∼ 3 ppm) PCBs. This dose also causes an increase in glucose uptake that is not seen at a higher dose (50 µM) suggesting that, at a lower dose, astrocytes are able to engage compensatory mechanisms to promote survival. Together, these data suggest that exposure to PCBs impact astrocytic metabolism, which is important to consider both in the context of human health and disease and in in vitro and in vivo disease models.

Low CD4+ cell count nadir exacerbates the impacts of APOE ε4 on functional connectivity and memory in adults with HIV.

OBJECTIVE: Nearly half of individuals living with HIV in the USA are now 50 or older. This rapidly ageing populace may be at an increasingly greater risk of Alzheimer's disease. However, the potential interaction between HIV-disease and Alzheimer's disease pathogenesis (i.e. Alzheimer's disease genetic risk factors) on brain function remains an open question. The present study aimed to investigate the impact of APOE ε4 on brain function in middle-aged to older people with HIV (PWH), as well as the putative interaction between ε4 and HIV disease severity. METHODS: Ninety-nine PWH participated in a cross-sectional study (56.3 ±â€Š6.5 years, range 41-70 years, 27 women, 26 ε4 carriers and 73 noncarriers). Structural MRI and resting-state functional MRI were collected to assess alterations in brain structure and functional connectivity, respectively. RESULTS: APOE ε4 was associated with worse memory performance and reduced functional connectivity in the memory network. The functional connectivity reduction was centred at the caudate nucleus rather than hippocampus and correlated with worse memory performance. In ε4 carriers, low CD4+ cell count nadir was associated with reduced functional connectivity in the memory network, but this association was absent in noncarriers. Furthermore, there was an indirect detrimental impact of ε4 on memory performance through memory network functional connectivity. However, this indirect effect was contingent on CD4+ cell count nadir, that is the indirect effect of ε4 on memory was only significant when CD4+ cell count nadir was low. INTERPRETATION: APOE ε4 is associated with reduced memory and reduced functional connectivity within the memory network, and low CD4+ cell count nadir -- indicating a history of severe immunosuppression -- may exacerbate the effects of ε4.

Cancer Chemotherapy Related Cognitive Impairment and the Impact of the Alzheimer's Disease Risk Factor APOE.

Cancer related cognitive impairment (CRCI) is a serious impairment to maintaining quality of life in cancer survivors. Cancer chemotherapy contributes to this condition through several potential mechanisms, including damage to the blood brain barrier, increases in oxidative stress and inflammation in the brain, and impaired neurogenesis, each of which lead to neuronal dysfunction. A genetic predisposition to CRCI is the E4 allele of the Apolipoprotein E gene (APOE), which is also the strongest genetic risk factor for Alzheimer's disease. In normal brains, APOE performs essential lipid transport functions. The APOE4 isoform has been linked to altered lipid binding, increased oxidative stress and inflammation, reduced turnover of neural progenitor cells, and impairment of the blood brain barrier. As chemotherapy also affects these processes, the influence of APOE4 on CRCI takes on great significance. This review outlines the main areas where APOE genotype could play a role in CRCI. Potential therapeutics based on APOE biology could mitigate these detrimental cognitive effects for those receiving chemotherapy, emphasizing that the APOE genotype could help in developing personalized cancer treatment regimens.

Benfotiamine and Cognitive Decline in Alzheimer's Disease: Results of a Randomized Placebo-Controlled Phase IIa Clinical Trial.

BACKGROUND: In preclinical models, benfotiamine efficiently ameliorates the clinical and biological pathologies that define Alzheimer's disease (AD) including impaired cognition, amyloid-ß plaques, neurofibrillary tangles, diminished glucose metabolism, oxidative stress, increased advanced glycation end products (AGE), and inflammation. OBJECTIVE: To collect preliminary data on feasibility, safety, and efficacy in individuals with amnestic mild cognitive impairment (aMCI) or mild dementia due to AD in a placebo-controlled trial of benfotiamine. METHODS: A twelve-month treatment with benfotiamine tested whether clinical decline would be delayed in the benfotiamine group compared to the placebo group. The primary clinical outcome was the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog). Secondary outcomes were the clinical dementia rating (CDR) score and fluorodeoxyglucose (FDG) uptake, measured with brain positron emission tomography (PET). Blood AGE were examined as an exploratory outcome. RESULTS: Participants were treated with benfotiamine (34) or placebo (36). Benfotiamine treatment was safe. The increase in ADAS-Cog was 43% lower in the benfotiamine group than in the placebo group, indicating less cognitive decline, and this effect was nearly statistically significant (p = 0.125). Worsening in CDR was 77% lower (p = 0.034) in the benfotiamine group compared to the placebo group, and this effect was stronger in the APOEɛ4 non-carriers. Benfotiamine significantly reduced increases in AGE (p = 0.044), and this effect was stronger in the APOEɛ4 non-carriers. Exploratory analysis derivation of an FDG PET pattern score showed a treatment effect at one year (p = 0.002). CONCLUSION: Oral benfotiamine is safe and potentially efficacious in improving cognitive outcomes among persons with MCI and mild AD.

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology.

APOE in the normal brain.

The APOE4 protein affects the primary neuropathological markers of Alzheimer's disease (AD): amyloid plaques, neurofibrillary tangles, and gliosis. These interactions have been investigated to understand the strong effect of APOE genotype on risk of AD. However, APOE genotype has strong effects on processes in normal brains, in the absence of the hallmarks of AD. We propose that CNS APOE is involved in processes in the normal brains that in later years apply specifically to processes of AD pathogenesis. We review the differences of the APOE protein found in the CNS compared to the plasma, including post-translational modifications (glycosylation, lipidation, multimer formation), focusing on ways that the common APOE isoforms differ from each other. We also review structural and functional studies of young human brains and control APOE knock-in mouse brains. These approaches demonstrate the effects of APOE genotype on microscopic neuron structure, gross brain structure, and behavior, primarily related to the hippocampal areas. By focusing on the effects of APOE genotype on normal brain function, approaches can be pursued to identify biomarkers of APOE dysfunction, to promote normal functions of the APOE4 isoform, and to prevent the accumulation of the pathologic hallmarks of AD with aging.

O-glycosylation on cerebrospinal fluid and plasma apolipoprotein E differs in the lipid-binding domain.

The O-glycoprotein apolipoprotein E (APOE), the strongest genetic risk factor for Alzheimer's disease, associates with lipoproteins. Cerebrospinal fluid (CSF) APOE binds only high-density lipoproteins (HDLs), while plasma APOE attaches to lipoproteins of diverse sizes with binding fine-tuned by the C-terminal loop. To better understand the O-glycosylation on this critical molecule and differences across tissues, we analyzed the O-glycosylation on APOE isolated from the plasma and CSF of aged individuals. Detailed LC-MS/MS analyses allowed the identification of the glycosite and the attached glycan and site occupancy for all detectable glycosites on APOE and further three-dimensional modeling of physiological glycoforms of APOE. APOE is O-glycosylated at several sites: Thr8, Thr18, Thr194, Ser197, Thr289, Ser290 and Ser296. Plasma APOE held more abundant (20.5%) N-terminal (Thr8) sialylated core 1 (Neu5Acα2-3Galß1-3GalNAcα1-) glycosylation compared to CSF APOE (0.1%). APOE was hinge domain glycosylated (Thr194 and Ser197) in both CSF (27.3%) and plasma (10.3%). CSF APOE held almost 10-fold more abundant C-terminal (Thr289, Ser290 and Ser296) glycosylation (36.8% of CSF peptide283-299 was glycosylated, 3.8% of plasma peptide283-299), with sialylated and disialylated (Neu5Acα2-3Galß1-3(Neu5Acα2-6) GalNAcα1-) core 1 structures. Modeling suggested that C-terminal glycosylation, particularly the branched disialylated structure, could interact across domains including the receptor-binding domain. These data, although limited by sample size, suggest that there are tissue-specific APOE glycoforms. Sialylated glycans, previously shown to improve HDL binding, are more abundant on the lipid-binding domain of CSF APOE and reduced in plasma APOE. This indicates that APOE glycosylation may be implicated in lipoprotein-binding flexibility.

Consumer preferences for beef with improved nutrient profile1.

Although beef is a nutrient-rich foodstuff excelling in protein, vitamins, and minerals, there is controversy regarding the fat content of beef and its healthfulness in the diet. Although much of the fatty acid (FA) content in beef is considered "healthy fats," many consumers are confused about the different classifications of FA. The objectives of this study were to determine consumers' knowledge about the nutritional value of beef and its importance in purchasing decisions, and to gain a better understanding about preferences for changes in FA composition. Objectives of the study were completed through 2 consumer studies: 1) an online survey and 2) a taste-panel auction. In the online survey, respondents were asked to choose between 2 steaks that varied in polyunsaturated and saturated FA levels, iron content, and price. Respondents were also asked to categorize "Monounsaturated Fat," "Polyunsaturated Fat," "Saturated Fat," and "Trans Fat," as either "healthy" or "unhealthy" both before and after an educational excerpt was provided. The results from the online survey indicated many consumers are unclear about the differences in beef nutritional value, specifically FA content. Initially, only 66.4%, 69.1%, 79.1%, and 79.2% of respondents correctly categorized the monounsaturated, polyunsaturated, saturated, and trans fat, respectively. However, more than 90% of respondents correctly categorized the various FAs after an educational excerpt was provided. After survey respondents better understood the healthfulness of FA in beef, they were also willing to pay a premium for a steak with improved FA composition. However, these premiums diminished when participants had to actually put forth a monetary value for a steak in the taste-panel auction. Research shows that there is variation among cattle for FA composition. This provides opportunity to identify cattle with a favorable composition and market this product to the increasing population of health-conscious consumers. Our results provide insight for beef promotion and marketing opportunities and indicate that relaying information about FA content is extremely important to collect a premium for healthier beef.

Histone deacetylase 6 inhibition rescues axonal transport impairments and prevents the neurotoxicity of HIV-1 envelope protein gp120.

Despite successful antiretroviral drug therapy, a subset of human immunodeficiency virus-1 (HIV)-positive individuals still display synaptodendritic simplifications and functional cognitive impairments referred to as HIV-associated neurocognitive disorders (HANDs). The neurological damage observed in HAND subjects can be experimentally reproduced by the HIV envelope protein gp120. However, the complete mechanism of gp120-mediated neurotoxicity is not entirely understood. Gp120 binds to neuronal microtubules and decreases the level of tubulin acetylation, suggesting that it may impair axonal transport. In this study, we utilized molecular and pharmacological approaches, in addition to microscopy, to examine the relationship between gp120-mediated tubulin deacetylation, axonal transport, and neuronal loss. Using primary rat cortical neurons, we show that gp120 decreases acetylation of tubulin and increases histone deacetylase 6 (HDAC6), a cytoplasmic enzyme that regulates tubulin deacetylation. We also demonstrate that the selective HDAC6 inhibitors tubacin and ACY-1215, which prevented gp120-mediated deacetylation of tubulin, inhibited the ability of gp120 to promote neurite shortening and cell death. We further observed by co-immunoprecipitation and confirmed with mass spectroscopy that exposure of neurons to gp120 decreases the association between tubulin and motor proteins, a well-established consequence of tubulin deacetylation. To assess the physiological consequences of this effect, we examined the axonal transport of brain-derived neurotrophic factor (BDNF). We report that gp120 decreases the velocity of BDNF transport, which was restored to baseline levels when neurons were exposed to HDAC6 inhibitors. Overall, our data suggest that gp120-mediated tubulin deacetylation causes impairment of axonal transport through alterations to the microtubule cytoskeleton.

Separation of Sialylated Glycan Isomers by Differential Mobility Spectrometry.

Mass spectrometry has proven itself to be an important technology for characterizing intact glycoproteins, glycopeptides, and released glycans. However, these molecules often present significant challenges during analysis. For example, glycans of identical molecular weights can be present in many isomeric forms, with one form having dramatically more biological activity than the others. Discriminating among these isomeric forms using mass spectrometry alone can be daunting, which is why orthogonal techniques, such as ion mobility spectrometry, have been explored. Here, we demonstrate the use of differential mobility spectrometry (DMS) to separate isomeric glycans differing only in the linkages of sialic acid groups (e.g., α 2,3 versus α 2,6). This ability extends from a small trisaccharide species to larger biantennary systems and is driven, in part, by the role of intramolecular solvation of the charge site(s) on these ions within the DMS environment.

Deciphering Isomers with a Multiple Reaction Monitoring Method for the Complete Detectable O-Glycan Repertoire of the Candidate Therapeutic, Lubricin.

Glycosylation is a fundamental post-translational modification, occurring on half of all proteins. Despite its significance, our understanding is limited, in part due to the inherent difficulty in studying these branched, multi-isomer structures. Accessible, detailed, and quantifiable methods for studying glycans, particularly O-glycans, are needed. Here we take a multiple reaction monitoring (MRM) approach to differentiate and relatively quantify all detectable glycans, including isomers, on the heavily O-glycosylated protein lubricin. Lubricin (proteoglycan 4) is essential for lubrication of the joint and eye. Given the therapeutic potential of lubricin, it is essential to understand its O-glycan repertoire in biological and recombinantly produced samples. O-Glycans were released by reductive ß-elimination and defined, showing a range of 26 neutral, sulfated, sialylated, and both sulfated and sialylated core 1 (Galß1-3GalNAcα1-) and core 2 (Galß1-3(GlcNAcß1-6)GalNAcα1-) structures. Isomer-specific MRM transitions allowed effective differentiation of neutral glycan isomers as well as sulfated isomeric structures, where the sulfate was retained on the fragment ions. This strategy was not as effective with labile sialylated structures; instead, it was observed that the optimal collision energy for the m/z 290.1 sialic acid B-fragment differed consistently between sialic acid isomers, allowing differentiation between isomers when fragmentation spectra were insufficient. This approach was also effective for purchased Neu5Acα2-3Galß1-4Glc and Neu5Acα2-6Galß1-4Glc and for Neu5Acα2-3Galß1-4GlcNAc and Neu5Acα2-6Galß1-4GlcNAc linkage isomers with the Neu5Acα2-6 consistently requiring more energy for optimal generation of the m/z 290.1 fragment. Overall, this method provides an effective and easily accessible approach for the quantification and annotation of complex released O-glycan samples.

Hydrogenolysis of Dinuclear PCNR Ligated PdII µ-Hydroxides and Their Mononuclear PdII Hydroxide Analogues.

The hydrogenolysis of mono- and dinuclear PdII hydroxides was investigated both experimentally and computationally. It was found that the dinuclear µ-hydroxide complexes {[(PCNR )Pd]2 (µ-OH)}(OTf) (PCNH =1-[3-[(di-tert-butylphosphino)methyl]phenyl]-1H-pyrazole; PCNMe =1-[3-[(di-tert-butylphosphino)methyl]phenyl]-5-methyl-1H-pyrazole) react with H2 to form the analogous dinuclear hydride species {[(PCNR )Pd]2 (µ-H)}(OTf). The dinuclear µ-hydride complexes were fully characterized, and are rare examples of structurally characterized unsupported singly bridged µ-H PdII dimers. The {[(PCNMe )Pd]2 (µ-OH)}(OTf) hydrogenolysis mechanism was investigated through experiments and computations. The hydrogenolysis of the mononuclear complex (PCNH )Pd-OH resulted in a mixed ligand dinuclear species [(PCNH )Pd](µ-H)[(PCC)Pd] (PCC=a dianionic version of PCNH bound through phosphorus P, aryl C, and pyrazole C atoms) generated from initial ligand "rollover" C-H activation. Further exposure to H2 yields the bisphosphine Pd0 complex Pd[(H)PCNH ]2 . When the ligand was protected at the pyrazole 5-position in the (PCNMe )Pd-OH complex, no hydride formed under the same conditions; the reaction proceeded directly to the bisphosphine Pd0 complex Pd[(H)PCNMe ]2 . Reaction mechanisms for the hydrogenolysis of the monomeric and dimeric hydroxides are proposed.