Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early‐infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P‐protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P‐protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss‐of‐functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.     

DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR‐activated murine macrophages

Delta‐like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR‐induced expression of key pro‐inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1‐deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN‐β and other pro‐inflammatory cytokines, including TNF‐α, IL‐12, and IL‐23. The expression of key proteins involved in IFN‐β signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL‐12 and IL‐23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS‐induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH‐mediated, pro‐inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.     

Clickable poly-l-lysine for the formation of biorecognition surfaces

Biomolecules are immobilized onto surfaces employing the fast and stable adsorption of poly-l-lysine (PLL) polymers and the versatile copper-free click chemistry reactions. This method provides the combined advantages of versatile surface adsorption with density control using polyelectrolytes and of the covalent and orthogonal immobilization of biomolecules with higher reaction rates and improved yields of click chemistry. Using DNA attachment as a proof of concept, control over the DNA probe density and applicability in electrochemical detection are presented.

The fast and stable adsorption of modified PLL on activated surfaces was combined with the versatile catalyst-free click chemistry for the fast and selective functionalization of substrates with DNA.     

Complete pathological responses in locally advanced rectal cancer after preoperative IMRT and integrated-boost chemoradiation

Strahlentherapie und Onkologie - To analyze the efficacy and safety of a new preoperative intensity-modulated radiotherapy (IMRT) and integrated-boost chemoradiation scheme. In all, 74 patients...     

Diagnosis and management of von Willebrand disease in Spain

The correct diagnosis and classification of Von Willebrand disease (VWD) is important for therapy and genetic counseling but is made difficult due to the variability of its clinical expression and limitations of laboratory methods. A national registry of VWD patients has been initiated in Spain. The results of a concise survey on the diagnosis of VWD show the frequency of VWD is fivefold greater in Spain than that expected from epidemiological studies in other European countries; this may result from overdiagnosis and/or a higher prevalence of VWD. These results clearly reinforce the need for the Spanish VWD registry. A consensus guideline for optimal treatment of VWD is being elaborated in Spain. Desmopressin (DDAVP) is the choice of treatment in responsive VWD patients. Von Willebrand factor concentrates (VWF/factor VIII) are used in individuals nonresponsive to DDAVP, when DDAVP is contraindicated, or in VWD types 2B and 3.     

Further evidence of olfactory ensheathing glia facilitating axonal regeneration after a complete spinal cord transection

Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT_2A receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.