Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics.

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

Hemoglobin A1c Trajectories During Pregnancy and Adverse Outcomes in Women With Type 2 Diabetes: A Danish National Population-Based Cohort Study.

OBJECTIVE: To identify and characterize groups of pregnant women with type 2 diabetes with distinct hemoglobin A1c (HbA1c) trajectories across gestation and to examine the association with adverse obstetric and perinatal outcomes. RESEARCH DESIGN AND METHODS: This was a retrospective Danish national cohort study including all singleton pregnancies in women with type 2 diabetes, giving birth to a liveborn infant, between 2004 and 2019. HbA1c trajectories were identified using latent class linear mixed-model analysis. Associations with adverse outcomes were examined with logistic regression models. RESULTS: A total of 1,129 pregnancies were included. Three HbA1c trajectory groups were identified and named according to the glycemic control in early pregnancy (good, 59%; moderate, 32%; and poor, 9%). According to the model, all groups attained an estimated HbA1c <6.5% (48 mmol/mol) during pregnancy, with no differences between groups in the 3rd trimester. Women with poor glycemic control in early pregnancy had lower odds of having an infant with large-for-gestational-age (LGA) birth weight (adjusted odds ratio [aOR] 0.57, 95% CI 0.40-0.83), and higher odds of having an infant with small-for-gestational age (SGA) birth weight (aOR 2.49, 95% CI 2.00-3.10) and congenital malformation (CM) (aOR 4.60 95% CI 3.39-6.26) compared with women with good glycemic control. There was no evidence of a difference in odds of preeclampsia, preterm birth, and caesarean section between groups. CONCLUSIONS: Women with poor glycemic control in early pregnancy have lower odds of having an infant with LGA birth weight, but higher odds of having an infant with SGA birth weight and CM.

Danish Diabetes Birth Registry 2: a study protocol of a national prospective cohort study to monitor outcomes of pregnancies of women with pre-existing diabetes.

INTRODUCTION: Despite technological developments and intensified care, pregnancies in women with pre-existing diabetes are still considered high-risk pregnancies. The rate of adverse outcomes in pregnancies affected by diabetes in Denmark is currently unknown, and there is a limited understanding of mechanisms contributing to this elevated risk. To address these gaps, the Danish Diabetes Birth Registry 2 (DDBR2) was established. The aims of this registry are to evaluate maternal and fetal-neonatal outcomes based on 5 years cohort data, and to identify pathophysiology and risk factors associated with short-term and long-term outcomes of pregnancies in women with pre-existing diabetes. METHODS AND ANALYSIS: The DDBR2 registry is a nationwide 5-year prospective cohort with an inclusion period from February 2023 to February 2028 of pregnancies in women with all types of pre-existing diabetes and includes registry, clinical and questionnaire data and biological samples of mother-partner-child trios. Eligible families (parents age ≥18 years and sufficient proficiency in Danish or English) can participate by either (1) basic level data obtained from medical records (mother and child) and questionnaires (partner) or (2) basic level data and additional data which includes questionnaires (mother and partner) and blood samples (all). The primary maternal outcome is Hemoglobin A1c (HbA1c) levels at the end of pregnancy and the primary offspring endpoint is the birth weight SD score. The DDBR2 registry will be complemented by genetic, epigenetic and metabolomic data as well as a biobank for future research, and the cohort will be followed through data from national databases to illuminate possible mechanisms that link maternal diabetes and other parental factors to a possible increased risk of adverse long-term child outcomes. ETHICS AND DISSEMINATION: Approval from the Ethical Committee is obtained (S-20220039). Findings will be sought published in international scientific journals and shared among the participating hospitals and policymakers. TRIAL REGISTRATION NUMBER: NCT05678543.

One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes.

Mass spectrometry (MS)-based proteomics workflows typically involve complex, multi-step processes, presenting challenges with sample losses, reproducibility, requiring substantial time and financial investments, and specialized skills. Here we introduce One-Tip, a proteomics methodology that seamlessly integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. One-Tip substantially simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells. The versatility of One-Tip is highlighted by nDIA identification of ~6000 proteins in single cells from early mouse embryos. Additionally, the study incorporates the Uno Single Cell Dispenser™, demonstrating the capability of One-Tip in single-cell proteomics with >3000 proteins identified per HeLa cell. We also extend One-Tip workflow to analysis of extracellular vesicles (EVs) extracted from blood plasma, demonstrating its high sensitivity by identifying >3000 proteins from 16 ng EV preparation. One-Tip expands capabilities of proteomics, offering greater depth and throughput across a range of sample types.

Seafloor primary production in a changing Arctic Ocean.

Phytoplankton and sea ice algae are traditionally considered to be the main primary producers in the Arctic Ocean. In this Perspective, we explore the importance of benthic primary producers (BPPs) encompassing microalgae, macroalgae, and seagrasses, which represent a poorly quantified source of Arctic marine primary production. Despite scarce observations, models predict that BPPs are widespread, colonizing ~3 million km2 of the extensive Arctic coastal and shelf seas. Using a synthesis of published data and a novel model, we estimate that BPPs currently contribute ~77 Tg C y-1 of primary production to the Arctic, equivalent to ~20 to 35% of annual phytoplankton production. Macroalgae contribute ~43 Tg C y-1, seagrasses contribute ~23 Tg C y-1, and microalgae-dominated shelf habitats contribute ~11 to 16 Tg C y-1. Since 2003, the Arctic seafloor area exposed to sunlight has increased by ~47,000 km2 y-1, expanding the realm of BPPs in a warming Arctic. Increased macrophyte abundance and productivity is expected along Arctic coastlines with continued ocean warming and sea ice loss. However, microalgal benthic primary production has increased in only a few shelf regions despite substantial sea ice loss over the past 20 y, as higher solar irradiance in the ice-free ocean is counterbalanced by reduced water transparency. This suggests complex impacts of climate change on Arctic light availability and marine primary production. Despite significant knowledge gaps on Arctic BPPs, their widespread presence and obvious contribution to coastal and shelf ecosystem production call for further investigation and for their inclusion in Arctic ecosystem models and carbon budgets.

Pre-analytical diagnostic differences despite high adherence to guidelines for gestational diabetes mellitus.

Regional variations in the prevalence of gestational diabetes mellitus (GDM) have been found across Denmark. The objectives of this exploratory survey were to evaluate adherence to the national guideline for screening and diagnosing GDM and to identify variations in pre-analytical or analytical factors, which could potentially contribute to variations in GDM prevalence across regions. In a national interview-based survey, obstetric departments and laboratories throughout Denmark handling GDM screening or diagnostic testing were invited to participate. Survey questionnaires were completed through personal interviews. In total, 21 of 22 identified obstetric departments and 44 of 45 identified laboratories participated. Adherence to guideline among obstetric departments ranged 67-100% and uniformity in laboratory procedures was high. However, the gestational age at the time of late diagnostic testing with oral glucose tolerance test (OGTT) varied considerably, with 48% (10/21) of departments testing outside the recommended 24-28 weeks' gestation. Procedural heterogeneity was most pronounced for the parts not described in current guidelines, with choice of laboratory equipment being the most diverse factor ranging 3-39% nationally. In conclusion, the overall adherence to the national guidelines was high across regions, and obstetric departments and laboratories had high uniformity in the procedures for screening and diagnosing GDM. Uniformity was generally high for procedures included in the guideline and low if not included. However, a high proportion of GDM testing was performed outside the recommended gestational window in late pregnancy, which may be a pre-analytical contributor to regional differences in GDM prevalence.

Living with schizophrenia and type 2 diabetes and the implication for diabetes self-care: A qualitative study.

AIM: To achieve an in-depth understanding of the challenges associated with diabetes management when having both schizophrenia and type 2 diabetes, while also identifying the needs for improved diabetes self-care. DESIGN: The study employed a qualitative explorative design utilizing a phenomenological-hermeneutic inspired approach, involving field observations and individual semistructured interviews. METHODS: Data were collected during 2020-2021 through 17 field observations of outpatient consultations and 13 individual semistructured interviews. Data, including field notes and verbatim transcribed interviews, underwent analysis following Ricoeur's interpretive philosophy, encompassing three levels: naïve reading, structural analysis and critical interpretation and discussion. This study adheres to the COREQ guidelines for qualitative research. RESULTS: Three key themes emerged: 'Diabetes when life is noisy', 'Sacrifices and compromises in life' and 'The double silence'. Everyday life is significantly affected when having both schizophrenia and T2D. The mental health state dominates in relation to diabetes self-care and individuals experience challenges balancing between the two conditions. However, there exists a general acknowledgement for diabetes and its long-term complications as a serious medical condition demanding careful attention and treatment. CONCLUSION: Self-managing two such complex conditions can be overwhelming and make it difficult for the individual to differentiate symptoms and prioritize diabetes care. Moreover, the existing fragmentation within healthcare systems poses communication challenges, resulting in disjointed patient pathways. IMPLICATIONS FOR PATIENT CARE: The study emphasizes the need for a holistic re that addresses the physical, emotional and social challenges. There is also a need for increased awareness and education among informal caregivers and healthcare professionals to foster better understanding and support.

Environmental drivers of Arctic communities based on metabarcoding of marine sediment eDNA.

Our ability to assess biodiversity at relevant spatial and temporal scales for informing management is of increasing importance given this is foundational to identify and mitigate the impacts of global change. Collecting baseline information and tracking ecological changes are particularly important for areas experiencing rapid changes and representing data gaps such as Arctic marine ecosystems. Environmental DNA has the potential to provide such data. We extracted environmental DNA from 90 surface sediment samples to assess eukaryote diversity around Greenland and Svalbard using two separate primer pairs amplifying different sections of the 18S rRNA gene. We detected 27 different phyla and 99 different orders and found that temperature and the change in temperature explained the most variation in the community in a single linear model, while latitude, sea ice cover and change in temperature explained the most variation in the community when assessed by individual non-linear models. We identified potential indicator taxa for Arctic climate change, including a terebellid annelid worm. In conclusion, our study demonstrates that environmental DNA offers a feasible method to assess biodiversity and identifies warming as a key driver of differences in biodiversity across these remote ecosystems.

Body mass index z-scores in the first 2 years of life were associated with adverse metabolic and anthropometric outcomes at 3 years of age.

AIM: We investigated associations between body mass index (BMI) z-scores for children aged 0-2 years and the BMI z-scores, body fat percentage and metabolic risk factors at 3 years of age. METHODS: This was a secondary analysis of the Lifestyle in Pregnancy and Offspring randomised controlled trial, carried out at two university hospitals in Denmark. It comprised 149 mothers with BMI ≥30 kg/m2 who did or did not receive a lifestyle intervention during pregnancy and a reference group of 97 mothers with normal-weight, with follow-up of their 3-year-old offspring. The children in these three groups were pooled for the data analyses, due to similar characteristics between groups. The BMI z-scores were calculated at 5 weeks, 5 months and 1, 2 and 3 years, using Danish reference groups. Their anthropometrics and metabolic outcomes were examined at 3 years of age. RESULTS: BMI z-scores at 5 months to 2 years were associated with BMI z-scores and body fat percentage at 3 years of age and BMI z-scores were not associated with metabolic risk factors at 3 years. CONCLUSION: BMI z-scores from 5 weeks of age were associated with adverse anthropometric outcomes but not with metabolic risk factors at 3 years of age.

Kidney Disease in Women With Previous Gestational Diabetes Mellitus: A Nationwide Register-Based Cohort Study.

OBJECTIVE: The association between gestational diabetes mellitus (GDM) and incident kidney disease, the mediating effects of diabetes and hypertension, and the impact of severity of metabolic dysfunction during pregnancy on the risk of incident kidney disease were investigated in this study. RESEARCH DESIGN AND METHODS: This Danish, nationwide, register-based cohort study included all women giving birth between 1997 and 2018. Outcomes included chronic kidney disease (CKD) and acute kidney disease, based on diagnosis codes. Cox regression analyses explored the association between GDM and kidney disease. A proxy for severity of metabolic dysfunction during pregnancy was based on GDM diagnosis and insulin treatment during GDM in pregnancy and was included in the models as an interaction term. The mediating effects of subsequent diabetes and hypertension prior to kidney disease were quantified using mediation analyses. RESULTS: Data from 697,622 women were used. Median follow-up was 11.9 years. GDM was associated with higher risk of CKD (adjusted hazard ratio [aHR] 1.92; 95% CI 1.67-2.21), whereas acute kidney disease was unrelated to GDM. The proportions of indirect effects of diabetes and hypertension on the association between GDM and CKD were 75.7% (95% CI 61.8-89.6) and 30.3% (95% CI 25.2-35.4), respectively, as assessed by mediation analyses. The CKD risk was significantly increased in women with insulin-treated GDM and no subsequent diabetes compared with women without GDM (aHR 2.35; 95% CI 1.39-3.97). CONCLUSIONS: The risk of CKD was significantly elevated after GDM irrespective of subsequent development of diabetes and hypertension. Furthermore, women with severe metabolic dysfunction during pregnancy had the highest CKD risk.

Characterization of Pt-coated twin paraboloidal laboratory capillary high energy X-ray optics.

Novel focusing optics composed of twin paraboloidal capillaries coated with Pt, for laboratory X-ray sources are presented and characterized. The optics are designed to focus the X-rays, resulting in an achromatic focused beam with photon energies up to 40 keV. The performance of the optics under different operational conditions is studied by comparing the energy-photon count spectra of the direct and focused beams. Based on these analyses, the optics gain and efficiency as a function of photon energy are determined. A focal spot of 8.5 µm with a divergence angle of 0.59° is observed. The obtained characteristics are discussed and related to theoretical considerations. Moreover, the suitability and advantages of the present optics for X-ray microdiffraction is demonstrated using polycrystalline aluminium. Finally, possibilities for further developments are suggested.

Global dataset of soil organic carbon in tidal marshes.

Tidal marshes store large amounts of organic carbon in their soils. Field data quantifying soil organic carbon (SOC) stocks provide an important resource for researchers, natural resource managers, and policy-makers working towards the protection, restoration, and valuation of these ecosystems. We collated a global dataset of tidal marsh soil organic carbon (MarSOC) from 99 studies that includes location, soil depth, site name, dry bulk density, SOC, and/or soil organic matter (SOM). The MarSOC dataset includes 17,454 data points from 2,329 unique locations, and 29 countries. We generated a general transfer function for the conversion of SOM to SOC. Using this data we estimated a median (± median absolute deviation) value of 79.2 ± 38.1 Mg SOC ha-1 in the top 30 cm and 231 ± 134 Mg SOC ha-1 in the top 1 m of tidal marsh soils globally. This data can serve as a basis for future work, and may contribute to incorporation of tidal marsh ecosystems into climate change mitigation and adaptation strategies and policies.

3D microscopy at the nanoscale reveals unexpected lattice rotations in deformed nickel.

In polycrystalline metals, plastic deformation is accompanied by lattice rotations resulting from dislocation glide. Following these rotations in three dimensions requires nondestructive methods that so far have been limited to grain sizes at the micrometer scale. We tracked the rotations of individual grains in nanograined nickel by using three-dimensional orientation mapping in a transmission electron microscope before and after in situ nanomechanical testing. Many of the larger-size grains underwent unexpected lattice rotations, which we attributed to a reversal of rotation during unloading. This inherent reversible rotation originated from a back stress-driven dislocation slip process that was more active for larger grains. These results provide insights into the fundamental deformation mechanisms of nanograined metals and will help to guide strategies for material design and engineering applications.

Health promotion intervention among women with recent gestational diabetes mellitus: penetration, participation, and baseline findings from the Face-it randomized controlled trial.

INTRODUCTION: Face-it is a randomized controlled trial for women with recent gestational diabetes mellitus (GDM) and their families designed to evaluate the effect of a health promotion intervention on type 2 diabetes mellitus (T2DM) risk and quality of life. This study examined (1) the penetration and participation rates for the Face-it trial, (2) the characteristics of the participating women and the potential differences in characteristics according to partner participation status, and (3) representativity of the women at baseline. RESEARCH DESIGN AND METHODS: We identified women with GDM during pregnancy and invited them and their partners to a baseline examination 10-14 weeks after delivery. Representativity was assessed by comparing the baseline participants with non-participating women, the general population of women with GDM delivering in Denmark, and populations from other intervention trials. RESULTS: The penetration rate was 38.0% (867/2279) and the participation rate was 32.9% (285/867). The 285 women who attended baseline had a mean age of 32.7 (±4.8) years and body mass index (BMI) of 28.1 (±5.4) kg/m2, and 69.8% had a partner who participated. The women participating with a partner were more often primiparous, born in Denmark (82.8% vs 68.2%), were younger, and more often had a BMI ≤24.9 kg/m2 (35.7% vs 21.2%) compared with women without a partner. Compared with the general population of women with GDM in Denmark, these women broadly had similar degree of heterogeneity, but had higher rates of primiparity and singleton deliveries, and lower rates of preterm delivery and prepregnancy obesity. CONCLUSIONS: The penetration and participation rates were acceptable. We found a high rate of partner participation. Overall, women participating with a partner were comparable with those participating without a partner. Participating women were broadly similar to the general national GDM population, however with prepregnancy obesity, multiparity, preterm delivery, and multiple pregnancy being less represented. TRIAL REGISTRATION NUMBER: NCT03997773.

Motivation for Behavior Change among Women with Recent Gestational Diabetes and Their Partners-A Qualitative Investigation among Participants in the Face-It Intervention.

Promoting diet and physical activity is important for women with recent gestational diabetes mellitus (GDM) and their partners to reduce the risk of future type 2 diabetes (T2D). The study aimed to understand how motivation for changing diet and physical activity behaviors among women with recent GDM and their partners was experienced after participation in the Danish Face-it intervention. Fourteen couples' interviews were conducted. Data analysis followed a reflexive thematic analysis. Guided by self-determination theory and interdependence theory, we identified four themes affecting couples' motivation for health behavior change: (1) The need to feel understood after delivery; (2) adjusting health expectations; (3) individual and mutual preferences for health behaviors; and (4) the health threat of future T2D as a cue to action. We found that couples in general perceived the Face-it intervention as useful and motivating. Using couple interviews increased our understanding of how the women and partners influenced each other's perspectives after a GDM-affected pregnancy and thus how targeting couples as opposed to women alone may motivate health behavior change.

Applying WHO2013 diagnostic criteria for gestational diabetes mellitus reveals currently untreated women at increased risk.

AIMS: To estimate the prevalence of gestational diabetes mellitus (GDM) in a Danish cohort comparing the current Danish versus the WHO2013 diagnostic criteria, and to evaluate adverse pregnancy outcomes among currently untreated women in the gap between the diagnostic thresholds. METHODS: Diagnostic testing was performed by a 75 g oral glucose tolerance test (OGTT) at 24-28 weeks' gestation in a cohort of pregnant women. GDM diagnosis was based on the current Danish criterion (2-h glucose ≥ 9.0 mmol/L, GDMDK) and on the WHO2013 criteria (fasting ≥ 5.1, 1 h ≥ 10.0 or 2 h glucose ≥ 8.5 mmol/L, GDMWHO2013). Currently untreated women fulfilling the WHO2013 but not the Danish diagnostic criteria were defined as New-GDM-women (GDMWHO2013-positive and GDMDK-negative). Adverse outcomes risks were calculated using logistic regression. RESULTS: OGTT was completed by 465 women at a median of 25.7 weeks' gestation. GDMDK prevalence was 2.2% (N = 10) and GDMWHO2013 21.5% (N = 100). New-GDM was present in 19.4% (N = 90), of whom 90.0% had elevated fasting glucose. Pregnancies complicated by New-GDM had higher frequencies of pregnancy-induced hypertension (13.3% vs 4.1%, p = 0.002), large-for-gestational-age infants (22.2% vs 9.9%, p = 0.004), neonatal hypoglycaemia (8.9% vs 1.9%, p = 0.004) and neonatal intensive care unit admission (16.7% vs 5.8%, p = 0.002) compared to pregnancies without GDM. CONCLUSIONS: GDM prevalence increased tenfold when applying WHO2013 criteria in a Danish population, mainly driven by higher fasting glucose levels. Untreated GDM in the gap between the current Danish and the WHO2013 diagnostic criteria resulted in higher risks of adverse pregnancy outcomes.

Prevalent fingerprint of marine macroalgae in arctic surface sediments.

Macroalgal forests export much of their production, partly supporting food webs and carbon stocks beyond their habitat, but evidence of their contribution in sediment carbon stocks is poor. We test the hypothesis that macroalgae contribute to carbon stocks in arctic marine sediments. We used environmental DNA (eDNA) fingerprinting on a large-scale set of surface sediment samples from Greenland and Svalbard. We evaluated eDNA results by comparing with traditional survey and tracer methods. The eDNA-based survey identified macroalgae in 94 % of the sediment samples covering shallow nearshore areas to 1460 m depth and 350 km offshore, with highest sequence abundance nearshore and with dominance of brown macroalgae. Overall, the eDNA results reflected the potential source communities of macroalgae and eelgrass assessed by traditional surveys, with the most abundant orders being common among different methods. A stable isotope analysis showed a considerable contribution from macroalgae in sediments although with high uncertainty, highlighting eDNA as a great improvement and supplement for documenting macroalgae as a contributor to sediment carbon stocks. Conclusively, we provide evidence for a prevalent contribution of macroalgal forests in arctic surface sediments, nearshore as well as offshore, identifying brown algae as main contributors.

The impact of inter-laboratory glucose bias on the diagnosis of gestational diabetes mellitus: Comparison of common automated central laboratory methods.

BACKGROUND AND AIMS: The diagnosis of gestational diabetes mellitus (GDM) is based exclusively on glucose measurements, which are highly influenced by pre-analytical and analytical factors. Therefore, poor agreement across laboratories may affect the prevalence of GDM. We aimed to determine the inter-laboratory bias of glucose measurements and the impact on GDM prevalence. MATERIAL AND METHODS: A prospective cohort study of women (n = 110) referred for second-trimester GDM diagnostics using a 75 g oral glucose tolerance test. Maternal glucose was assessed from venous plasma at fasting, 1 h and 2 h. Venous blood were collected in Fluoride Citrate tubes and frozen. Samples were analyzed at five central laboratories using four different automated glucose Hexokinase methods and GDM prevalence was evaluated according to WHO2013 diagnostic criteria. RESULTS: Maximum inter-laboratory bias was 0.19, 0.30 and 0.27 mmol/L in fasting, 1 h and 2 h samples, respectively. GDM prevalence ranged 30.0-41.1% across laboratories. CONCLUSION: Inter-laboratory bias for mean venous glucose was low and within desirable limits. Nonetheless, the impact on GDM prevalence was considerable, which may inappropriately affect clinical practice.