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.

Healthcare Professionals' Knowledge of and Attitudes Towards the Use of Time in Range in Diabetes Management: Online Survey Across Seven Countries.

INTRODUCTION: Time in range (TIR) is a metric of glycaemic target management derived from continuous glucose monitoring (CGM) data. This study aimed to understand knowledge of and attitudes towards use of TIR among healthcare professionals (HCPs), and gain insights into benefits and barriers to its use in clinical practice. METHODS: An online survey was disseminated across seven countries. Participants were sampled from online HCP panels and were aware of TIR (defined as amount of time in, below, and above target range). Participants were HCPs classified as specialists (SP), generalists (GP), or allied HCPs (AP; diabetes nurse specialists, diabetes educators, general nurses, nurse practitioners/physician assistants). RESULTS: Respondents included 741 SP, 671 GP and 307 AP. Most HCPs (approximately 90%) agreed TIR is likely/somewhat likely to become the standard of diabetes management. Perceived benefits of TIR included helping to optimise medication regimen (SP, 71%; GP, 73%; AP, 74%), giving HCPs the knowledge and insights to make informed clinical decisions (SP, 66%; GP, 61%; AP, 72%), and empowering people with diabetes with information to successfully manage their diabetes (SP, 69%; GP, 77%; AP, 78%). Barriers to wider adoption included limited CGM access (SP, 65%; GP, 74%; AP, 69%) and lack of HCP training/education (SP, 45%; GP, 59%; AP, 51%). Most participants considered integration of TIR into clinical guidelines, recognition of TIR by regulators as a primary clinical endpoint, and recognition of TIR by payers as a parameter for diabetes treatment evaluation as key factors for increased use. CONCLUSIONS: Overall, HCPs agreed on the benefits of using TIR for diabetes management. Besides raising awareness among HCPs and people with diabetes, more training and healthcare system updates are needed to facilitate increased TIR use. In addition, integration into clinical guidelines and recognition by regulators and payers are needed.

'Time in range' is the proportion of time in a day that a person's glucose level is within a particular range. The purpose of this study was to understand knowledge of and attitudes towards use of TIR among healthcare professionals. The study was carried out using an online survey and participants from seven countries were included. Participants were healthcare professionals classified as specialists (SP), generalists (GP), or allied healthcare professionals (AP; diabetes nurse specialists, diabetes educators, general nurses, nurse practitioners, or physician assistants). Overall, 1719 participants were included in the study. Most healthcare professionals (approximately 90%) agreed that time in range is likely/somewhat likely to become the standard of diabetes management. Participants reported the following benefits of time in range: helping to optimise medication regimen, giving healthcare professionals the knowledge and insights to make informed clinical decisions, and empowering people with diabetes with information to successfully manage their diabetes. The most common barrier to wider time in range adoption was limited access to continuous glucose monitoring (SP, 65%; GP, 74%; AP, 69%), followed by lack of healthcare professionals' training/education (SP, 45%; GP, 59%; AP, 51%). Most participants considered integration of time in range into clinical guidelines, recognition of time in range by regulators as a primary clinical endpoint, and recognition of time in range by payers as a parameter for evaluation of diabetes treatment as key factors for the increased use of time in range.

Effect of a biosynthetic bacterial 6-phytase on the digestibility of phosphorus and phytate in midlactating dairy cows.

The effect of a biosynthetic bacterial 6-phytase (PhyG) on the digestibility and excretion of crude protein (CP), phosphorus (P), and phytate-P (PP) in midlactating dairy cows was investigated. Thirty Holstein-Friesians were assigned to three treatments with 10 cows per treatment in a randomized block design. Cows were fed forage (grass and corn silage) provided ad libitum, and a concentrate (without added inorganic phosphate) administered separately in amounts individualized per cow according to milk production, supplemented with phytase according to treatment. The formulated forage-to-concentrate-ratio was ~65%:35%. Dietary treatments comprised the control diet (CON) and CON supplemented with 2,000 (PhyG2,000) or 5,000 (PhyG5,000) phytase units (FTU)/kg DM in the total diet. The experiment comprised an 18-d preperiod for the collection of data to facilitate the allocation of cows to the treatments, followed by a 19-d experimental period comprising a 14-d diet adaptation period and 5 d of twice daily feces collection. Fecal samples were analyzed for the determination of apparent total tract digestibility (ATTD) of chemical constituents in the diet. The ATTD of PP was 92.6% in CON suggesting a high but incomplete degradation of phytate by ruminal microbial phytases. Cows fed PhyG2,000 exhibited increased ATTD of CP and PP [68.4% (2.7% points above CON) and 95.1% (2.5% points above CON), respectively] whilst PhyG5,000 further increased ATTD PP and also increased ATTD P [54.1% (7.8% points above CON)]; ATTD of Ca tended to be increased in PhyG5,000 vs. CON. Linear dose-response relationships were observed for ATTD of DM, CP, P, Ca, and PP. In addition, fecal excretion of P, and PP linearly reduced and that of Ca and CP tended to linearly reduce with increasing PhyG dose level. No difference was observed for DM intake and milk composition was unaffected except for milk protein which tended to be higher in cows fed PhyG5,000 than CON. In summary, the addition of exogenous phytase at 2,000 FTU/kg or higher to diets of lactating dairy cows improved P, PP, Ca, and CP digestibility and reduced fecal excretion of P, PP, and CP in a dose-dependent manner.

Traditionally, it has been believed that dairy cows are able to fully utilize the phosphorus (P) in feed, including that from plant-derived phytate, because of phytase activity of bacteria in the rumen. However, recent data have shown otherwise. This study investigated the effect of a biosynthetic bacterial 6-phytase supplemented to the diets of midlactating dairy cows on the digestibility and excretion of phosphorus and other key nutrients, over a 19-d experimental period. The experimental diets were commercially relevant in composition and low in phosphorus. At either or both of two tested dose levels (2,000 and 5,000 phytase units (FTU) per kilogram DM in the total diet), the exogenous phytase increased the digestibility and reduced fecal excretion of crude protein (CP), total P, and phytate-P compared with a comparable unsupplemented diet. The increases in CP, PP, and P digestibility were phytase-dose dependent. In addition, at the highest dose level, the phytase tended to increase the protein content of milk. The findings indicate that the use of exogenous phytase can improve P and protein utilization in dairy cows and offers an important approach to optimizing nutrient balance and reducing environmental P and nitrogen (N) pollution from dairy farms.

The association between second trimester ultrasound fetal biometrics and gestational diabetes.

OBJECTIVE: Gestational diabetes mellitus (GDM) is the most common metabolic complication of pregnancy. The incidence of GDM is increasing worldwide and 5-25% of pregnancies are diagnosed with GDM depending on screening strategies and diagnostic criteria. GDM may lead to obstetric complications and increases the risk of adult metabolic disease in the offspring. Timely identification of GDM allows for regulation of maternal glucose levels which may reduce the obstetric complications considerably. The aim of this study is to investigate the association between second trimester ultrasound biometrics and GDM. STUDY DESIGN: This is a retrospective cohort study including 2697 singleton pregnancies attending second trimester ultrasound scan at 20 + 0 to 20 + 6 weeks' gestation and giving birth at Aalborg University Hospital in the year 2020. Ultrasound measurements included head circumference (HC), abdominal circumference (AC), femur length (FL) and estimated fetal weight (EFW) by Hadlock's formula. Women with pregestational diabetes were excluded. GDM screening was performed on indication using oral-glucose-tolerance-test (OGTT) including 75 g glucose and a 2-hour serum glucose value ≥ 9 mmol/L was considered diagnostic. The association between fetal biometrics and GDM was investigated by logistic regression. RESULTS: A total of 174 (6.5 %) were diagnosed with GDM. The incidence of GDM in pregnancies with biometrics above the 90th centile was; FL: 10.5 %, HC: 8.8 %, AC: 7.6 %, EFW: 9.3 %. Fetal biometrics above the 90th centile was significantly associated with GDM; ORFL = 2.07, p = 0.001; ORHC = 1.89, p = 0.001; ORAC = 1.63, p = 0.033; OREFW = 1.64, p = 0.036. This association remained significant for HC and FL when adjusted for maternal obesity (Body Mass Index ≥ 27): ORHC(adj)=1.56, p = 0.019; ORFL(adj) = 1.57, p = 0.049. CONCLUSION: At the second trimester scan, fetal biometrics above the 90th centile increase the risk of GDM. In pregnancies that are later diagnosed with GDM fetal growth is increased already at the second trimester scan. Such knowledge underlines the importance of early identification of GDM.

Effect of two phytases at two doses on performance and phytate degradation in broilers during 1-21 days of age.

The effect of two microbial phytases at two dose-levels on performance and apparent ileal digestibility (AID) of nutrients in broilers fed European-type diets was studied. A total of 1,200 d-old Ross 308 male broilers were randomly assigned to 5 treatments with 30 birds/pen and 8 pens/treatment. A nutritionally adequate positive control (PC) diet was tested against 4 experimental diets containing reduced total P, retainable P, Ca and Na as per the recommended nutritional contribution for Buttiauxella phytase (Phy B) at 1,000 FTU/kg (-1.87 g/kg, -1.59 g/kg, -1.99 g/kg and -0.4 g/kg vs. PC, respectively). Experimental diets were supplemented with Phy B at 500 FTU/kg or 1,000 FTU/kg, or Citrobacter phytase (Phy C) at 1,000 FTU/kg or 2,000 FTU/kg. Diets were based on corn, soybean meal, rapeseed meal and sunflower meal and formulated by phase (starter 1-10 d, grower 11-21 d) in crumbled or pelleted form. Overall (d 1-21), at 1,000 FTU/kg, birds fed Phy C exhibited lower BWG (-2.7%), FI (-3.4%) and tibia ash (-2.2%) vs. PC (P < 0.05), and reduced BWG (-3.6%), FI (-3.9%) and tibia ash (-1.8%) vs. Phy B (P < 0.05). Phy B at 1,000 FTU/kg and Phy C at 2,000 FTU/kg maintained performance equivalent to the PC. Digestibility of Ca did not differ among phytase treatments but at 1,000 FTU/kg AID P was greater with Phy B than Phy C (72.3% vs. 62.7%, P < 0.05). Ileal phytate (myo-inositol hexakisphosphate, IP6) digestibility was greatest with Phy B at 1,000 FTU/kg which was higher than Phy C at 1,000 FTU/kg (87.6 vs. 60.6%, P < 0.05). The findings indicate a higher phytate degradation rate of Phy B than Phy C at equivalent dose-level and this is correlated to the performance of the broilers.

Corrigendum to "Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to weaned piglets fed a corn-soybean meal-based diet without added inorganic phosphate" [Animal Nutrition 6/1 (2020) 24-30].

[This corrects the article DOI: 10.1016/j.aninu.2019.11.003.].

Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to weaned piglets fed a corn-soybean meal-based diet without added inorganic phosphate.

The utility of a next generation biosynthetic bacterial 6-phytase (PhyG) in restoring bone ash, bone phosphorus (P) content and performance in piglets depleted in P was evaluated. A total of 9 treatments were tested as follows. Treatment 1, a negative control (NC) diet; treatments 2, 3, 4, NC supplemented with 250, 500 or 1,000 FTU/kg of PhyG; treatments 5, 6, NC supplemented with 500 or 1,000 FTU/kg of a commercial Buttiauxella sp phytase (PhyB); treatments 7, 8, 9, NC supplemented with monocalcium phosphate (MCP) to provide 0.7, 1.4 and 1.8 g/kg digestible P, equating to a digestible P content of 1.8, 2.5 and 2.9 g/kg. The latter constituting the positive control (PC) diet with adequate P and calcium (Ca). The NC was formulated without inorganic P (1.1 g digestible P/kg) and reduced in Ca (5.0 g/kg). Additional limestone was added to treatments 7 to 9 to maintain Ca-to-P ratio between 1.2 and 1.3. A total of 162 crossed Pietrain × (Large White × Landrace) 21-d-old piglets (50% males and 50% females) were fed adaptation diets until 42 d old and then assigned to pens with 2 pigs/pen and 9 pens/treatment in a completely randomized block design. Piglets were fed mash diets based on corn and soybean meal ad libitum for 28 d. At the end of the study, one piglet perpen was euthanized and the right feet collected for determination of bone strength, bone ash and mineral content. Compared with the PC, the NC group had reduced average daily gain (ADG) and increased feed conversion ratio (FCR) during all growth phases and overall, and at d 28 (70 d old) NC pigs had bones with reduced ash, Ca and P content (P < 0.05). The PhyG at 250 FTU/kg improved bone ash vs. NC. Increasing PhyG dose linearly or quadratically improved bone ash, ADG and FCR (P < 0.05). At ≥ 500 FTU/kg, both PhyG and PhyB maintained ADG and FCR equivalent to PC. Linear regression analysis was done to compare the measured response parameters to increasing digestible P from MCP. Based on this analysis it was shown that PhyG and PhyB at 1,000 FTU/kg could replace 1.83 and 1.66 g/kg digestible P from MCP in the diet, respectively, on average across metacarpi bone ash, ADG or FCR. These findings suggest that the biosynthetic phytase is highly effective in the tested dietary setting.

Pancreatic exocrine insufficiency in diabetes mellitus - prevalence and characteristics.

BACKGROUND: The prevalence of pancreatic exocrine insufficiency (PEI) in diabetes mellitus (DM) varies widely between studies, which may be explained by methodological problems. We aimed to establish the prevalence of PEI in DM using the faecal elastase-1 (FE-1) assay as a screening test, and to further investigate these patients by the mixed 13C-triglyceride (13C-MTG) breath test. METHODS: One hundred and thirty-three consecutive type 1 or type 2 DM patients without known exocrine pancreatic disorders were recruited. Demographic parameters, stool consistency, stool frequency, routine laboratory tests, and the presence of DM complications were registered. An FE-1 value <200 µg/g was used as the screening cut-off for PEI, and patients with FE-1 values below this level were referred for a 13C-MTG breath test. RESULTS: One hundred and two patients returned faecal samples. The prevalence of PEI as measured by low FE-1 was 13%. Insulin usage, type 1 DM, and DM duration were associated with low FE-1. Stool habits were unaffected by low FE-1. Twelve out of 13 patients with low FE-1 performed the breath test, which was normal in all cases. CONCLUSIONS: The prevalence of PEI defined by FE-1 was low in our mixed cohort of type 1 and 2 DM patients. Furthermore, there was a discrepancy between FE-1 and the breath test. Hence, the role of FE-1 in evaluating pancreatic exocrine function in DM should be evaluated in larger studies in order to clarify the association between low FE-1 and clinically relevant PEI.

MicroRNA Profiling in the Medial and Lateral Habenula of Rats Exposed to the Learned Helplessness Paradigm: Candidate Biomarkers for Susceptibility and Resilience to Inescapable Shock.

Depression is a highly heterogeneous disorder presumably caused by a combination of several factors ultimately causing the pathological condition. The genetic liability model of depression is likely to be of polygenic heterogeneity. miRNAs can regulate multiple genes simultaneously and therefore are candidates that align with this model. The habenula has been linked to depression in both clinical and animal studies, shifting interest towards this region as a neural substrate in depression. The goal of the present study was to search for alterations in miRNA expression levels in the medial and lateral habenula of rats exposed to the learned helplessness (LH) rat model of depression. Ten miRNAs showed significant alterations associating with their response to the LH paradigm. Of these, six and four miRNAs were significantly regulated in the MHb and LHb, respectively. In the MHb we identified miR-490, miR-291a-3p, MiR-467a, miR-216a, miR-18b, and miR-302a. In the LHb miR-543, miR-367, miR-467c, and miR-760-5p were significantly regulated. A target gene analysis showed that several of the target genes are involved in MAPK signaling, neutrophin signaling, and ErbB signaling, indicating that neurotransmission is affected in the habenula as a consequence of exposure to the LH paradigm.

Do Gender-Specific and High-Resolution Three Dimensional Body Charts Facilitate the Communication of Pain for Women? A Quantitative and Qualitative Study.

BACKGROUND: Chronic pain is more prevalent among women; however, the majority of standardized pain drawings are often collected using male-like androgynous body representations. OBJECTIVE: The purpose of this study was to assess whether gender-specific and high-resolution three-dimensional (3D) body charts facilitate the communication of pain for women. METHODS: Using mixed-methods and a cross-over design, female patients with chronic pain were asked to provide detailed drawings of their current pain on masculine and feminine two-dimensional (2D) body schemas (N=41, Part I) or on female 2D and 3D high-resolution body schemas (N=41, Part II) on a computer tablet. The consistency of the drawings between body charts were assessed by intraclass correlation coefficient (ICC) and Bland-Altman plots. Semistructured interviews and a preference questionnaire were then used to obtain qualitative and quantitative responses of the drawing experience. RESULTS: The consistency between body charts were high (Part I: ICC=0.980, Part II: ICC=0.994). The preference ratio for the masculine to feminine body schemas were 6:35 and 18:23 for the 2D to 3D female body charts. Patients reported that the 3D body chart enabled a more accurate expression of their pain due to the detailed contours of the musculature and bone structure, however, patients also reported the 3D body chart was too human and believed that skin-like appearance limited 'deep pain' expressions. CONCLUSIONS: Providing gender-specific body charts may facilitate the communication of pain and the level of detail (2D vs 3D body charts) should be used according to patients' needs.

Presynaptic plasticity as a hallmark of rat stress susceptibility and antidepressant response.

Two main questions are important for understanding and treating affective disorders: why are certain individuals susceptible or resilient to stress, and what are the features of treatment response and resistance? To address these questions, we used a chronic mild stress (CMS) rat model of depression. When exposed to stress, a fraction of rats develops anhedonic-like behavior, a core symptom of major depression, while another subgroup of rats is resilient to CMS. Furthermore, the anhedonic-like state is reversed in about half the animals in response to chronic escitalopram treatment (responders), while the remaining animals are resistant (non-responder animals). Electrophysiology in hippocampal brain slices was used to identify a synaptic hallmark characterizing these groups of animals. Presynaptic properties were investigated at GABAergic synapses onto single dentate gyrus granule cells. Stress-susceptible rats displayed a reduced probability of GABA release judged by an altered paired-pulse ratio of evoked inhibitory postsynaptic currents (IPSCs) (1.48 ± 0.25) compared with control (0.81 ± 0.05) and stress-resilient rats (0.78 ± 0.03). Spontaneous IPSCs (sIPSCs) occurred less frequently in stress-susceptible rats compared with control and resilient rats. Finally, a subset of stress-susceptible rats responding to selective serotonin reuptake inhibitor (SSRI) treatment showed a normalization of the paired-pulse ratio (0.73 ± 0.06) whereas non-responder rats showed no normalization (1.2 ± 0.2). No changes in the number of parvalbumin-positive interneurons were observed. Thus, we provide evidence for a distinct GABAergic synaptopathy which associates closely with stress-susceptibility and treatment-resistance in an animal model of depression.

Molecular profiling of the lateral habenula in a rat model of depression.

OBJECTIVE: This study systematically investigated the effect of chronic mild stress and response to antidepressant treatment in the lateral habenula at the whole genome level. METHODS: Rat whole genome expression chips (Affymetrix) were used to detect gene expression regulations in the lateral habenula of rats subjected to chronic mild stress (mild stressors exchanged twice a day for 8 weeks). Some rats received antidepressant treatment during fifth to eights week of CMS. The lateral habenula gene expression profile was studied through the gene ontology and signal pathway analyses using bioinformatics. Real-time quantitative polymerase chain reaction (RT-PCR) was used to verify the microarray results and determine the expression of the Fcrla, Eif3k, Sec3l1, Ubr5, Abca8a, Ankrd49, Cyp2j10, Frs3, Syn2, and Znf503 genes in the lateral habenula tissue. RESULTS: In particular we found that stress and antidepressant treatment affected intracellular cascades like growth factor receptor signaling, G-protein-coupled receptor signaling, and Wnt signaling - processes involved in the neuroplastic changes observed during the progression of depression and antidepressant treatment. CONCLUSION: The present study suggests an important role of the lateral habenula in the development of depression-like conditions and correlates to previous studies demonstrating a significant role of the lateral habenula in depressive-like conditions and antidepressant treatment.

Predicting transition temperatures of elastin-like polypeptide fusion proteins.

Elastin-like polypeptides (ELPs) are thermally sensitive peptide polymers that undergo thermally triggered phase separation and this behavior is imparted to soluble proteins when they are fused to an ELP. The transition temperature of the ELP fusion protein is observed to be different than that of a free ELP, indicating that the surface properties of the fused protein modulate the thermal behavior of ELPs. Understanding this effect is important for the rational design of applications that exploit the phase transition behavior of ELP fusion proteins. We had previously developed a biophysical model that explained the effect of hydrophobic proteins on depressing the transition temperature of ELP fusion proteins relative to free ELP. Here, we extend the model to elucidate the effect of hydrophilic proteins on the thermal behavior of ELP fusion proteins. A linear correlation was found between overall residue composition of accessible protein surface weighted by a characteristic transition temperature for each residue and the difference in transition temperatures between the ELP protein fusion and the corresponding free ELP. In breaking down the contribution of residues to polar, nonpolar, and charged, the model revealed that charged residues are the most important parameter in altering the transition temperature of an ELP fusion relative to the free ELP.

Vesicular signalling and immune modulation as hedonic fingerprints: proteomic profiling in the chronic mild stress depression model.

Extensive preclinical research has focused at unravelling the underlying molecular mechanisms leading to depression and recovery. In this study, we investigated the quantitative changes in protein abundance in the ventral hippocampal granular cell layer. We compared different phenotypes from the chronic mild stress (CMS) model of depression using chronic administration with two selective serotonin reuptake inhibitors (SSRIs), escitalopram and sertraline. We isolated granular cells using Laser-Capture Microdissection (LCM) and we identified their regulated proteins using two-dimensional (2D) differential gel electrophoresis (DIGE) and tandem mass spectrometry (MS/MS). The majority of the proteins we identified were enzymes involved in different metabolic activities. Additional proteins were functionally classified as vesicular proteins and immune system proteins. Rab GDP dissociation inhibitor alpha (GDIA) and syntaxin-binding protein 1 (STXB1) were potential markers for stress reactivity. Dynamin 1 (DYN1), glutathione S-transferase omega-1 (GSTO1) and peroxiredoxin (PRDX6) were associated with treatment response. In addition, an imbalance between different post-translationally modified versions of DYN1 and GSTO1 potentially accounted for SSRI treatment refraction. In the present study, we searched for new markers of stress reactivity and treatment response as well as any underlying molecular mechanisms correlating to the development of anhedonia and antidepressant therapy refraction. Our results pointed towards an essential role of post-translational modifications in both vesicular and immune protein systems.

Low maternal care exacerbates adult stress susceptibility in the chronic mild stress rat model of depression.

In the present study we report the finding that the quality of maternal care, in early life, increased the susceptibility to stress exposure in adulthood, when rats were exposed to the chronic mild stress paradigm. Our results indicate that high, as opposed to low maternal care, predisposed rats to a differential stress-coping ability. Thus rats fostered by low maternal care dams became more prone to adopt a stress-susceptible phenotype developing an anhedonic-like condition. Moreover, low maternal care offspring had lower weight gain and lower locomotion, with no additive effect of stress. Subchronic exposure to chronic mild stress induced an increase in faecal corticosterone metabolites, which was only significant in rats from low maternal care dams. Examination of glucocorticoid receptor exon 17 promoter methylation in unchallenged adult, maternally characterized rats, showed an insignificant tendency towards higher total cytosine methylation in rats from low maternal care dams. Assessment of methylation in the resilient versus anhedonic-like rat phenotypes, revealed only minor differences. Thus, maternal care status seems to be a strong predictor or trait marker for the behavioural phenotype.

Synergistic antidepressant-like action of gaboxadol and escitalopram.

According to current theories on the etiopathogenesis and pathophysiology of depression, both GABAergic and monoaminergic transmitter systems are perturbed. Consequently, the present study addressed the putative antidepressant action of the sedative-hypnotic GABAA receptor agonist, gaboxadol, separately and in combination with the selective serotonin reuptake inhibitor (SSRI) escitalopram. The rat chronic mild stress model was used to test the chronic antidepressant properties of gaboxadol in this depression model. Sucrose intake used as a read-out on anhedonic-like behavior indicated that the drug response rate for gaboxadol (5 mg/kg/day, i.p.) was similar to that measured for escitalopram (5 mg/kg/day, i.p.), however, the rate increased when the two drugs were co-administered, suggesting a synergistic action. Using in vivo electrophysiological recordings in dorsal raphe nucleus (DRN) of anesthetised rats, the present results showed that one week treatment with gaboxadol (5 mg/kg/day, i.p.) or with escitalopram (5 mg/kg/day, i.p.), followed by a 24 h washout period, did not affect DRN 5-HT neuronal firing per se, but in rats treated with both drugs for one week, the firing rate of DRN 5-HT neurons was significantly increased. Immunohistochemical estimations of cell proliferation in the hippocampal dentate gyrus did not reveal any effect of gaboxadol on chronic mild stressed rats, indicating that neurogenesis per se is not systematically associated with recovery from anhedonic-like behavior. Taken together, our data reveal for the first time an antidepressant action of gaboxadol and indicate a synergistic mechanism, regarding rapid onset of action and efficacy, when co-administered with escitalopram.

Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry.

Bacterial Protein A (PrtA) and Protein G (PrtG) are widely used for affinity purification of antibodies. An understanding of how PrtA and PrtG bind to different isotypes of immunoglobulin type G (IgG) and to their corresponding Fc fragments is essential for the development of PrtA and PrtG mimetic ligands and for the establishment of generic processes for the purification of various antibodies. In this paper, the interactions between the two IgG-binding proteins and IgG of two different subclasses, IgG1 and IgG4, as well as their analogous Fc fragments have been studied by isothermal titration calorimetry. The results indicate that both protein ligands bind IgG and Fc fragments strongly with Ka values in the range of 10(7) -10(8) M(-1) and for both ligands, the interaction with both IgG isotypes is enthalpically driven though entropically unfavorable. Moreover, variation in the standard entropic and standard enthalpic contribution to binding between the two isotypes as well as between IgG and Fc fragment implies that the specific interaction with PrtA varies according to IgG isotype. In contrast to PrtA, PrtG bound to F(ab')(2) fragment with a Ka value of 5.1 × 10(5) M(-1) ; thus underscoring the usefulness of PrtA as a preferred ligand for generic antibody purification processes.

Comparison of plasma metanephrines measured by a commercial immunoassay and urinary catecholamines in the diagnosis of pheochromocytoma.

BACKGROUND: The diagnosis of pheochromocytomas requires consideration among patients suffering from hypertension, unexplained spells, incidental adrenal masses, or a family history of pheochromocytoma. Accordingly, the diagnosis requires a biochemical test with high sensitivity and specificity. AIM: To compare plasma free metanephrines as measured by a commercial immunoassay and the 24-hour urinary excretion of catecholamines. METHOD: Plasma free metanephrines were measured in 185 patients suspected of pheochromocytoma. Concomitant measurements of urinary catecholamines were performed in 115 patients. Based on clinical findings, imaging and biochemistry 11 cases were found; 9 were diagnosed with pheochromocytoma, one patient with paraganglioma and one patient with ganglioneuroma. RESULTS: All patients with pheochromocytoma/paraganglioma had abnormally elevated concentrations of either plasma metanephrine or normetanephrine. The patient with ganglioneuroma had normal plasma metanephrine levels, corresponding to a sensitivity of 91%. In two patients where pheochromocytoma was excluded, plasma metanephrin or normetanephrine was above the reference level, corresponding to a specificity of 99%. Urinary catecholamines were determined in 10 of 11 patients with a positive diagnosis, and all 10 showed elevated levels of either urinary epinephrine or norepinephrine, including the patient with ganglioneuroma (equivalent to a sensitivity of 100%). Seven patients, in whom pheochromocytoma was excluded, had elevated urinary catecholamines (equivalent to a specificity of 94%). CONCLUSION: Measurement of plasma free metanephrines by immunoassay appears to be a useful diagnostic test in patients suspected of pheochromocytoma, with a high specificity as compared with urinary catecholamines. The latter may result in fewer false-positive findings, an outcome which may be particularly troublesome.

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation.

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R(h)): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R(h): 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.