Maternal and fetal outcomes in pregnancy complicated by pre-existing lupus nephritis: Insights from a developing country Pakistan.

BACKGROUND: Pregnancy in women with systemic lupus erythematosus (SLE) has remained a great challenge for clinicians in terms of maternal and fetal outcomes. The outcomes in women with pre-existing lupus nephritis (LN) are variable. The impact of different classes of LN on maternal and fetal outcomes during pregnancy is not well defined, as data is very scarce, especially from the developing countries. METHODS: A retrospective analysis was conducted on 52 women with 89 pregnancies. All had biopsy-proven LN. Those women who conceived at least 6 months after the diagnosis were included. The analysis was conducted between July 1998 and June 2018 at Sindh Institute of Urology and Transplantation (SIUT), evaluating the outcomes for both the mother and the fetus with a minimum follow-up of 12 months after child birth. RESULTS: The mean maternal age at SLE diagnosis was 21.45 ± 6 years and at first pregnancy was 26.49 ± 5.63 years. The mean disease duration was 14.02 ± 19.8 months. At conception, 47 (52.8%) women were hypertensive, 9 (10%) had active disease while 38 (42.7%) and 42 (47.2%) were in complete and partial remission, respectively. A total of 17 (19.1%) were on mycophenolate mofetil (MMF), which was switched to azathioprine (AZA). Out of 89 pregnancies, 56 (62.9%) were successful, while 33 (37.07%) had fetal complications like spontaneous abortion, stillbirth, perinatal death, and intrauterine growth retardation (IUGR). There were more vaginal deliveries (33 [58.92%]) than caesarean sections (23 [41.07%]). Renal flare was observed in 33 (37.1%) women while 15 (16.9%) developed pre-eclampsia. Proliferative LN was found in 56 (62.9%) cases, but no significant differences were found in maternal and fetal outcomes in relation to LN classes (p = .58). However, disease outcomes at 12 months were significantly poor in those with active disease at the time of conception (p < .05). There was only one maternal death. A total of 10 (11.2%) women showed deterioration in renal function and 5 (5.6%) were dialysis-dependent at 12 months. CONCLUSION: The maternal and fetal outcomes in pre-existing LN depend on the disease activity at the time of conception. No correlation was found between International Society of Nephrology/Renal Pathology Society (ISN/RPS) classes of LN and adverse disease and pregnancy outcomes.

Interventions Based on Behavior Change Techniques to Encourage Physical Activity or Decrease Sedentary Behavior in Community-Dwelling Adults Aged 50-70: Systematic Review With Intervention Component Analysis.

Increasing physical activity (PA) and/or decreasing sedentary behaviors is important in the delay and prevention of long-term conditions. PA can help maintain function and independence and decrease the need for hospitalization/institutionalization. Activity rates often decline in later life resulting in a need for interventions that encourage uptake and adherence through the use of Behavior Change Techniques (BCTs). We conducted a systematic review of the evidence for interventions that included BCTs in community-dwelling adults with a mean age of 50-70. The review followed PRISMA guidelines. The interventions were psychosocial, nonpharmacological, and noninvasive interventions utilizing components based on BCTs that evaluated change in PA and/or sedentary behavior. Intervention Component Analysis (ICA) was used to synthesize effectiveness of intervention components. Twelve randomized controlled trials were included in this review. The mean sample age was 50-64. Thirteen BCTs were used across all studies, and the most commonly used techniques were goals and planning, feedback and monitoring, and natural consequences. Seven intervention components linked with BCTs were found: personalized goal setting, tailored feedback from facilitators, on-site and postintervention support, education materials and resources, reinforcing change on behavior and attitudes, self-reported monitoring, and social connectedness. All components, except for social connectedness, were associated with improved health behavior and PA levels. The interventions that use BCTs have incorporated strategies that reinforce change in behavior and attitudes toward PA.

Proteomics-Based Mapping of Bronchopulmonary Dysplasia-Associated Changes in Noninvasively Accessible Oral Secretions.

OBJECTIVE: To determine if oral secretions (OS) can be used as a noninvasively collected body fluid, in lieu of tracheal aspirates (TA), to track respiratory status and predict bronchopulmonary dysplasia (BPD) development in infants born <32 weeks. STUDY DESIGN: This was a retrospective, single center cohort study that included data and convenience samples from week-of-life (WoL) 3 from 2 independent preterm infant cohorts. Using previously banked samples, we applied our sample-sparing, high-throughput proteomics technology to compare OS and TA proteomes in infants born <32 weeks admitted to the Neonatal Intensive Care Unit (NICU) (Cohort 1; n = 23 infants). In a separate similar cohort, we mapped the BPD-associated changes in the OS proteome (Cohort 2; n = 17 infants including 8 with BPD). RESULTS: In samples collected during the first month of life, we identified 607 proteins unique to OS, 327 proteins unique to TA, and 687 overlapping proteins belonging to pathways involved in immune effector processes, neutrophil degranulation, leukocyte mediated immunity, and metabolic processes. Furthermore, we identified 37 OS proteins that showed significantly differential abundance between BPD cases and controls: 13 were associated with metabolic and immune dysregulation, 10 of which (eg, SERPINC1, CSTA, BPI) have been linked to BPD or other prematurity-related lung disease based on blood or TA investigations, but not OS. CONCLUSIONS: OS are a noninvasive, easily accessible alternative to TA and amenable to high-throughput proteomic analysis in preterm newborns. OS samples hold promise to yield actionable biomarkers of BPD development, particularly for prospective categorization and timely tailored treatment of at-risk infants with novel therapies.

The Use of Digital Health by South Asian Communities: Scoping Review.

BACKGROUND: South Asian individuals experience a higher burden of chronic diseases and limited access to health care services compared with their Caucasian peers. Digital health interventions can enhance the delivery of health care, minimize health inequities, and consequently improve health status among minority ethnic groups. However, it is unclear how South Asian people view and perceive the use of digital health technologies to support their health needs. OBJECTIVE: The aim of the review is to identify South Asian individuals' experiences and attitudes of digital health and explore the barriers and facilitators affecting their use of digital health services. METHODS: The Arksey and O'Malley methodological framework was used to guide this scoping review. Five electronic databases were examined for pertinent papers, which were augmented by searching bibliographies of the retrieved papers and gray literature. A total of 1328 potentially relevant papers were retrieved from the initial search, and the supplemental search added 7 papers to the final list of potentially included papers. Each paper on the initial inclusion list was independently reviewed, leaving 15 papers to be included in the review. RESULTS: Data were analyzed thematically leading to the development of two overarching themes: (1) barriers to uptake of digital health and (2) facilitators of use of digital health services. There was a general consensus that South Asian communities still struggle with inadequate access to digital health technologies. Some studies suggest multiple initiatives to improve accessibility and acceptability of digital health services within South Asian communities in order to mitigate health disparities and develop a more inclusive health care system. These include the development of multiple-language and culturally sensitive interventions and digital skill development sessions. Most studies were conducted in South Asian countries, focusing on measurable outcomes of digital health interventions. Few explored the experiences and views of South Asian community members residing in the West as a minority ethnic group, for example, British South Asians. CONCLUSIONS: Literature mapping proposes that South Asian people frequently struggle with a health care system that may limit their access to digital health services, and sometimes fails to consider social and cultural needs. There is growing evidence that digital health interventions have the potential to facilitate supported self-management, which is part of the plans to adopt person-centered care. These interventions are particularly important for overcoming some of the challenges, for example, time constraints, safety, and gender sensitivity, associated with the delivery of health care interventions in minority ethnic groups such as South Asians in the United Kingdom, and thus to improve minority ethnic groups' access to health care services to support individual health needs, and consequently enhance health status.

Protein kinases PknA and PknB independently and coordinately regulate essential Mycobacterium tuberculosis physiologies and antimicrobial susceptibility.

The Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB are essential for growth and have been proposed as possible drug targets. We used a titratable conditional depletion system to investigate the functions of these kinases. Depletion of PknA or PknB or both kinases resulted in growth arrest, shortening of cells, and time-dependent loss of acid-fast staining with a concomitant decrease in mycolate synthesis and accumulation of trehalose monomycolate. Depletion of PknA and/or PknB resulted in markedly increased susceptibility to ß-lactam antibiotics, and to the key tuberculosis drug rifampin. Phosphoproteomic analysis showed extensive changes in protein phosphorylation in response to PknA depletion and comparatively fewer changes with PknB depletion. These results identify candidate substrates of each kinase and suggest specific and coordinate roles for PknA and PknB in regulating multiple essential physiologies. These findings support these kinases as targets for new antituberculosis drugs and provide a valuable resource for targeted investigation of mechanisms by which protein phosphorylation regulates pathways required for growth and virulence in M. tuberculosis.

Using plasma proteomics to investigate viral infections of the central nervous system including patients with HIV-associated neurocognitive disorders.

State-of-the-art liquid chromatography/mass spectrometry (LC/MS)-based proteomic technologies, using microliter amounts of patient plasma, can detect and quantify several hundred plasma proteins in a high throughput fashion, allowing for the discovery of clinically relevant protein biomarkers and insights into the underlying pathobiological processes. Using such an in-house developed high throughput plasma proteomics allowed us to identify and quantify > 400 plasmas proteins in 15 min per sample, i.e., a throughput of 100 samples/day. We demonstrated the clinical applicability of our method in this pilot study by mapping the plasma proteomes from patients infected with human immunodeficiency virus (HIV) or herpes virus, both groups with involvement of the central nervous system (CNS). We found significant disease-specific differences in the plasma proteomes. The most notable difference was a decrease in the levels of several coagulation-associated proteins in HIV vs. herpes virus, among other dysregulated biological pathways providing insight into the differential pathophysiology of HIV compared to herpes virus infection. In a subsequent analysis, we found several plasma proteins associated with immunity and metabolism to differentiate patients with HIV-associated neurocognitive disorders (HAND) compared to cognitively normal people with HIV (PWH), suggesting the presence of plasma-based biomarkers to distinguishing HAND from cognitively normal PWH. Overall, our high-throughput plasma proteomics pipeline enables the identification of distinct proteomic signatures of HIV and herpes virus, which may help illuminate divergent pathophysiology behind virus-associated neurological disorders.

The impact of COVID-19 lockdowns on physical activity amongst older adults: evidence from longitudinal data in the UK.

BACKGROUND: A sedentary lifestyle increases the risk of adverse health outcomes and frailty,particularly for older adults. To reduce transmission during the COVID-19 pandemic, people were instructed to stay at home, group sports were suspended, and gyms were closed, thereby limiting opportunities for physical activity. Whilst evidence suggests that physical activity levels reduced during the pandemic, it is unclear whether the proportion of older adults realising the recommended minimum level of physical activity changed throughout the various stages of lockdown. METHODS: We used a large sample of 3,660 older adults (aged ≥ 65) who took part in the UK Household Longitudinal Study's annual and COVID-19 studies. We examined changes in the proportion of older adults who were realising the UK Chief Medical Officers' physical activity recommendations for health maintenance at several time points before and after COVID-19 lockdowns were imposed. We stratified these trends by the presence of health conditions, age, neighbourhood deprivation, and pre-pandemic activity levels. RESULTS: There was a marked decline in older adults' physical activity levels during the third national lockdown in January 2021. The proportion realising the Chief Medical Officers' physical activity recommendations decreased from 43% in September 2020 to 33% in January 2021. This decrease in physical activity occurred regardless of health condition, age, neighbourhood deprivation, or pre-pandemic activity levels. Those doing the least activity pre-lockdown increased their activity during lockdowns and those doing the most decreased their activity levels. CONCLUSIONS: Reductions in older adults' physical activity levels during COVID-19 lockdowns have put them at risk of becoming deconditioned and developing adverse health outcomes. Resources should be allocated to promote the uptake of physical activity in older adults to reverse the effects of deconditioning.

Women's perceptions of PERSPECTIVE: a breast cancer risk stratification e-platform.

BACKGROUND: Breast cancer risk stratification categorizes a woman's potential risk of developing the disease as near-population, intermediate, or high. In accordance, screening and follow up for breast cancer can readily be tailored following risk assessment. Recent efforts have focussed on developing more accessible means to convey this information to women. This study sought to document the relevance of an informational e-platform developed for these purposes. OBJECTIVE: To begin to assess a newly developed breast cancer risk stratification and decision support e-platform called PERSPECTIVE (PErsonalised Risk Stratification for Prevention and Early deteCTIon of breast cancer) among women who do not know their personal breast cancer risk (Phase 1). Changes (pre- and post- e-platform exposure) in knowledge of breast cancer risk and interest in undergoing genetic testing were assessed in addition to perceptions of platform usability and acceptability. METHODS: Using a pre-post design, women (N = 156) of differing literacy and education levels, aged 30 to 60, with no previous breast cancer diagnosis were recruited from the general population and completed self-report e-questionnaires. RESULTS: Mean e-platform viewing time was 18.67 min (SD 0.65) with the most frequently visited pages being breast cancer-related risk factors and risk assessment. Post-exposure, participants reported  significantly higher breast cancer-related knowledge (p < .001). Increases in knowledge relating to obesity, alcohol, breast density, menstruation, and the risk estimation process remained even when sociodemographic variables age and education were controlled. There were no significant changes in genetic testing interest post-exposure. Mean ratings for e-platform acceptability and usability were high: 26.19 out of 30 (SD 0.157) and 42.85 out of 50 (SD 0.267), respectively. CONCLUSIONS: An informative breast cancer risk stratification e-platform targeting healthy women in the general population can significantly increase knowledge as well as support decisions around breast cancer risk and assessment. Currently underway, Phase 2, called PERSPECTIVE, is seeking further content integration and broader implementation .

Sample Preparation for High-Throughput Urine Proteomics Using 96-Well Polyvinylidene Fluoride (PVDF) Membranes.

Proteomics analysis of urine samples allows for studying the impact of system perturbation. However, meaningful proteomics-based biomarker discovery projects often require the analysis of large patient cohorts with hundreds of samples to describe the biological variability. Thus, robust high-throughput sample processing methods are a prerequisite for clinical proteomics pipelines that minimize experimental bias due to individual sample processing methods. Herein we describe a high-throughput method for parallel 96-well plate-based processing of urine samples for subsequent LC/MS-based proteomic analyses. Protein digestion and subsequent sample processing steps are efficiently performed in 96-well polyvinylidene fluoride (PVDF) membrane plate allowing for the use of vacuum manifolds for rapid liquid transfer, and multichannel pipettes and/or liquid handing robots. In this chapter we make available a detailed step-by-step protocol for our 'MStern blotting' sample processing strategy applied to patient urine samples followed by mass spectrometry-based proteomics analysis. Subsequently, we provide an example application using minimal volume of urine samples (e.g. 150 µL) collected from children pre and post thoracotomy to identify the predominant sites of protein catabolism and aid in the design of therapies to ameliorate protein catabolism and breakdown during critical illness. Furthermore, we demonstrate how the systemic state is reflected in the urine as an easily obtainable, stable, and safe biofluid.

Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis.

BACKGROUND: Genetic mutations in α-actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known. METHODS: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-ß levels increase phosphorylation of ACTN4. RESULTS: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-ß stimulates phosphorylation of ACTN4 at S159 in podocytes. CONCLUSIONS: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.

Commissioning social care for people with dementia living at home: Findings from a national survey.

OBJECTIVE: To explore the complexities, circumstances, and range of services commissioned for people with dementia living at home. METHODS: A national survey was used to collect data from English local authorities in 2015. Commissioners of services for older adults were invited to complete a questionnaire. An exploratory cluster analysis of nominal data was conducted using a TwoStep procedure to identify distinct groups. RESULTS: A total of 122 authorities (83%) responded to the request. Four approaches to commissioning were identified, reflecting commissioning practices at the organisational, strategic, and individual service user levels. Commissioning at the service user level was most apparent. Bivariate analysis found that these configurations were not associated with the types of dementia specific services provided but were related to the number available. Authorities delivered a greater range of specialist services when joint commissioning between social care and health partners was undertaken. However, the joint commissioning of services was less observed in services specifically for people with dementia than in generic services for all older people. There was limited evidence that local circumstances (population configuration and deprivation levels) were associated with this approach to commissioning. CONCLUSIONS: The significant role of health partners in the delivery of social care services to support older people living with dementia in their own homes is evident. As the population with dementia ages and physical health needs increase, how dementia specific services differ from and complement those services available to all older people warrants further investigation.

Avoir sa santé en main : le sentiment d'habilitation tel que perçu par les jeunes adultes souffrant d'un cancer avancé.

CONTEXTE: Le sentiment d'habilitation sur sa santé (health related empowerment) est un concept fondamental des soins centrés sur la personne. Toutefois, on en sait peu sur la manière dont ce concept s'articule chez les jeunes adultes ayant un diagnostic de cancer avancé. OBJECTIF: Explorer le sentiment d'habilitation à la lumière des expériences de soins de santé vécues par les jeunes adultes en phase de cancer avancé. CADRE ET PARTICIPANTS: Douze jeunes adultes (âgés de 21 à 39 ans) ont été recrutés dans un grand centre de cancérologie de Montréal, au Québec. MÉTHODOLOGIE: Des entretiens en profondeur durant entre 36 et 90 minutes ont été menés individuellement, enregistrés et retranscrits mot pour mot, puis analysés par thèmes. RÉSULTATS: Tout au long de l'expérience du cancer, les participants ont témoigné du désir soutenu de participer activement à leur traitement et à leurs soins. Quatre thèmes sont ressortis des données décrivant les processus d'attente, de prise en charge de la maladie, de mise en action et de recadrage. Sous-jacents à ces thèmes se trouvent les notions de conscience du corps, les obstacles à surmonter pour obtenir des soins, l'optimisation de la santé et la réflexion sur l'héritage qu'on laisse derrière soi. CONCLUSIONS: De façon générale, les participants voulaient demeurer en contrôle de la situation malgré les multiples difficultés inhérentes à un cancer de stade avancé. Si elles sont corroborées par d'autres recherches, ces conclusions pourraient orienter les approches de soins en oncologie afin qu'elles soient véritablement adaptées aux besoins des jeunes adultes.

Taking control over our health: Empowerment as perceived by young adults living with advanced cancer.

BACKGROUND: Health-related empowerment is a key concept in person-centred care. However, little is known of its core elements in young adults diagnosed with advanced cancer. OBJECTIVE: To explore empowerment in the context of young adults' healthcare experiences who are now in advanced stages of cancer. SETTING & PARTICIPANTS: Twelve young adults (aged 21 to 39 years) were recruited from a large cancer centre in Montreal, Quebec. METHODS: In-depth interviews lasting between 36 and 90 minutes were conducted individually, audio-recorded, transcribed verbatim, and analyzed using thematic analysis. RESULTS: Throughout the cancer trajectory, participants reported a sustained desire to be actively involved in their treatment and care. Four themes emerged from the data representing processes of waiting, managing, acting, and revisiting. Subsumed under these were notions of body ownership, facing obstacles to care, optimizing health, and (re)considering their legacy. CONCLUSIONS: Overall, participants wanted to remain in control of their situation despite the multiple challenges related to advanced cancer. If corroborated further, these findings should inform supportive cancer care approaches that are truly tailored to the needs of young adults.

Co-regulation proteomics reveals substrates and mechanisms of APC/C-dependent degradation.

Using multiplexed quantitative proteomics, we analyzed cell cycle-dependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C(CDH1)-dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.

Specialist services in early diagnosis and support for older people with dementia in England: Staff roles and service mix.

OBJECTIVES: This study investigated staff roles and tasks in Community Mental Health Teams (CMHT) and memory clinics, which are provided within a framework determined by local Clinical Commissioning Groups. METHODS: A cross-sectional survey design was used to collect data in England in 2015. Teams were identified by mental health providers (n = 68) and invited to complete a questionnaire. RESULTS: Fifty-one NHS Trusts responded to the request. The response rate varied. Data were obtained for all Clinical Commissioning Groups areas in 3 of the 9 regions in England, but only half in one of them. CMHTs were significantly more likely to have larger staff groups. Compared with memory clinics they were also more likely to have staff that were not professionally qualified. The occupational therapist role showed a strong association with the provision of all services in CMHTs. Both CMHTs and memory clinics provided information and advice about dementia. CMHTs provided more services associated with the support of a person with dementia at home. CONCLUSION: Variations in the staff mix in CMHTs and memory clinics reflected their different functions. There was limited evidence in both of profession specific interventions relating to the provision of support, information, therapy and education, associated with either diagnosis or long-term support. The potential for a single service to undertake both diagnostic and long-term support and the associated costs and benefits are areas for future research.

Aqueous solvation from the water perspective.

The response of water re-solvating a charge-transfer dye (deprotonated Coumarin 343) after photoexcitation has been measured by means of transient THz spectroscopy. Two steps of increasing THz absorption are observed, a first ∼10 ps step on the time scale of Debye relaxation of bulk water and a much slower step on a 3.9 ns time scale, the latter of which reflecting heating of the bulk solution upon electronic relaxation of the dye molecules from the S1 back into the S0 state. As an additional reference experiment, the hydroxyl vibration of water has been excited directly by a short IR pulse, establishing that the THz signal measures an elevated temperature within ∼1 ps. This result shows that the first step upon dye excitation (10 ps) is not limited by the response time of the THz signal; it rather reflects the reorientation of water molecules in the solvation layer. The apparent discrepancy between the relatively slow reorientation time and the general notion that water is among the fastest solvents with a solvation time in the sub-picosecond regime is discussed. Furthermore, non-equilibrium molecular dynamics simulations have been performed, revealing a close-to-quantitative agreement with experiment, which allows one to disentangle the contribution of heating to the overall THz response from that of water orientation.

Adjuvant-induced Human Monocyte Secretome Profiles Reveal Adjuvant- and Age-specific Protein Signatures.

Adjuvants boost vaccine responses, enhancing protective immunity against infections that are most common among the very young. Many adjuvants activate innate immunity, some via Toll-Like Receptors (TLRs), whose activities varies with age. Accordingly, characterization of age-specific adjuvant-induced immune responses may inform rational adjuvant design targeting vulnerable populations. In this study, we employed proteomics to characterize the adjuvant-induced changes of secretomes from human newborn and adult monocytes in response to Alum, the most commonly used adjuvant in licensed vaccines; Monophosphoryl Lipid A (MPLA), a TLR4-activating adjuvant component of a licensed Human Papilloma Virus vaccine; and R848 an imidazoquinoline TLR7/8 agonist that is a candidate adjuvant for early life vaccines. Monocytes were incubated in vitro for 24 h with vehicle, Alum, MPLA, or R848 and supernatants collected for proteomic analysis employing liquid chromatography-mass spectrometry (LC-MS) (data available via ProteomeXchange, ID PXD003534). 1894 non-redundant proteins were identified, of which ∼30 - 40% were common to all treatment conditions and ∼5% were treatment-specific. Adjuvant-stimulated secretome profiles, as identified by cluster analyses of over-represented proteins, varied with age and adjuvant type. Adjuvants, especially Alum, activated multiple innate immune pathways as assessed by functional enrichment analyses. Release of lactoferrin, pentraxin 3, and matrix metalloproteinase-9 was confirmed in newborn and adult whole blood and blood monocytes stimulated with adjuvants alone or adjuvanted licensed vaccines with distinct clinical reactogenicity profiles. MPLA-induced adult monocyte secretome profiles correlated in silico with transcriptome profiles induced in adults immunized with the MPLA-adjuvanted RTS,S malaria vaccine (Mosquirix™). Overall, adjuvants such as Alum, MPLA and R848 give rise to distinct and age-specific monocyte secretome profiles, paralleling responses to adjuvant-containing vaccines in vivo Age-specific in vitro modeling coupled with proteomics may provide fresh insight into the ontogeny of adjuvant action thereby informing targeted adjuvanted vaccine development for distinct age groups.

Abundance-based classifier for the prediction of mass spectrometric peptide detectability upon enrichment (PPA).

The function of a large percentage of proteins is modulated by post-translational modifications (PTMs). Currently, mass spectrometry (MS) is the only proteome-wide technology that can identify PTMs. Unfortunately, the inability to detect a PTM by MS is not proof that the modification is not present. The detectability of peptides varies significantly making MS potentially blind to a large fraction of peptides. Learning from published algorithms that generally focus on predicting the most detectable peptides we developed a tool that incorporates protein abundance into the peptide prediction algorithm with the aim to determine the detectability of every peptide within a protein. We tested our tool, "Peptide Prediction with Abundance" (PPA), on in-house acquired as well as published data sets from other groups acquired on different instrument platforms. Incorporation of protein abundance into the prediction allows us to assess not only the detectability of all peptides but also whether a peptide of interest is likely to become detectable upon enrichment. We validated the ability of our tool to predict changes in protein detectability with a dilution series of 31 purified proteins at several different concentrations. PPA predicted the concentration dependent peptide detectability in 78% of the cases correctly, demonstrating its utility for predicting the protein enrichment needed to observe a peptide of interest in targeted experiments. This is especially important in the analysis of PTMs. PPA is available as a web-based or executable package that can work with generally applicable defaults or retrained from a pilot MS data set.

MStern Blotting-High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates.

We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification.