Pain response to vaccination in newborn infants of diabetic mothers.

BACKGROUND: Response to pain is altered in infants who were exposed to pain- and stressful events in the neonatal period. Infants of diabetic mothers receive several heel sticks after birth for measuring blood glucose and thus may show changes in their behavioral and physiologic response to pain. Moreover, maternal hyperglycemia may alter activity of the hypothalamic pituitary adrenal (HPA) axis reactivity. STUDY DESIGN: In total, 43 infants of diabetic mothers and 30 control infants were included into the study. Response to pain was assessed at 3 months of age following two intramuscular injections for vaccination. We assessed behavioral (Bernese pain scale), physiologic (heart rate) and hormonal (salivary cortisol) pain response to vaccination as well as spinal sensitization (flexion withdrawal reflex). RESULTS: Infants of diabetic mothers received a median number of 5 [4-19] painful events compared to 1 [1-3] in the control group. Heart rate reactivity differed significantly between groups. Infants of diabetic mothers had higher peaks (p = 0.002) and needed more time to recover to baseline (p < 0.001). Moreover, infants of diabetic mothers showed higher peak cortisol (p = 0.001) and a higher relative cortisol increase (p = 0.015). Flexor withdrawal reflex thresholds were significantly lower in infants of diabetic mothers (p = 0.003). CONCLUSION: The increase of physiologic and hormonal responses to pain in infants of diabetic mothers is probably caused by repeated painful events and an altered metabolic profile.

Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila.

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.

Mycophenolate mofetil prevents the delayed T cell response after pilocarpine-induced status epilepticus in mice.

Growing clinical and laboratory evidence corroborates a role for the immune system in the pathophysiology of epilepsy. In order to delineate the immune response following pilocarpine-induced status epilepticus (SE) in the mouse, we monitored the kinetics of leukocyte presence in the hippocampus over the period of four weeks. SE was induced following a ramping protocol of pilocarpine injection into 4-5 weeks old C57BL/6 mice. Brains were removed at days 1-4, 14 or 28 after SE, and the hippocampi were analyzed via flow cytometry, via quantitative reverse transcriptase PCR (qRT-PCR) and via immunohistochemistry. Epileptogenesis was confirmed by Timm staining of mossy fiber sprouting in the inner molecular layer of the dentate gyrus. The flow cytometry data revealed a biphasic immune response following pilocarpine-induced SE with a transient increase in activated CD11b+ and F4/80+ macrophages within the first four days replaced by an increase in CD3+ T-lymphocytes around day 28. This delayed T cell response was confirmed via qRT-PCR and via immunohistochemistry. In addition, qRT-PCR data could show that the delayed T cell response was associated with an increased CD8/CD4 ratio indicating a cytotoxic T cell response after SE. Intriguingly, early intervention with mycophenolate mofetil administration on days 0-3 after SE prevented this delayed T cell response. These results show an orchestrated immunological sequela and provide evidence that the delayed T cell response is sensitive to early immunomodulatory intervention.

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila.

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRSL262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

Cell-selective labelling of proteomes in Drosophila melanogaster.

The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRS(LtoG)) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through 'click chemistry'. To test these methods for applicability in vivo, we expressed MetRS(LtoG) cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms.

Outcome of extremely low gestational age newborns after introduction of a revised protocol to assist preterm infants in their transition to extrauterine life.

AIM: To evaluate the outcome of a cohort of extremely low gestational age newborn infants (ELGAN) below 26-week gestation who were treated following a revised, gentle delivery room protocol to assist them in the transition and adaptation to extrauterine life. METHODS: A cohort of infants with a gestational age (GA) below 26 weeks (study group; n = 164) was treated according to a revised delivery room protocol. The protocol included an optimized prenatal management, strict use of continuous positive airway pressure (CPAP), avoiding mechanical ventilation and early administration of surfactant without intubation. The parameters management of respiratory distress syndrome, survival, neonatal morbidity and neurodevelopmental outcome were compared with a historical control group (n = 44). RESULTS: Seventy-four per cent of the study group infants were initially treated with CPAP and surfactant administration without intubation. In comparison with the control group, significantly less children were intubated in the delivery room (24% vs. 41%) and needed mechanical ventilation (51% vs. 72%; both p < 0.05). Furthermore, compared with the historical control overall mortality (20% vs. 39%), rate of bronchopulmonary dysplasia (18% vs. 37%) and IVH > II° (10% vs. 33%) in survivors were significantly lower during the observational period (all p < 0.05). Neurodevelopmental outcome was normal in 70% of examined study group infants. CONCLUSIONS: A revised delivery room management protocol was applied safely to infants with a GA below 26 completed weeks with improved rates of survival and morbidity.