The mhp gene cluster from Escherichia coli constitutes a model system to study bacterial degradation of 3-(3-hydroxyphenyl)propionic acid (3HPP). In this work the regulation of the inducible mhp catabolic genes has been studied by genetic and biochemical approaches. The Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively, show a peculiar arrangement leading to transcripts that are complementary at their 5 -ends. By using Pr-lacZ and Pa-lacZ translational fusions and gel retardation assays, we have shown that the mhpR gene product behaves as a 3HPP-dependent activator of the Pa promoter, being the expression from Pr constitutive and MhpR-independent. DNase I footprinting experiments and mutational analysis mapped an MhpR-protected region, centered at position ؊58 with respect to the Pa transcription start site, which is indispensable for MhpR binding and in vivo activation of the Pa promoter. Superimposed in the specific MhpR-mediated regulation of the Pa promoter, we have observed a strict catabolite repression control carried out by the cAMP receptor protein (CRP) that allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available and 3HPP is present in the medium. Gel retardation assays revealed that the specific activator, MhpR, is essential for the binding of the second activator, CRP, to the Pa promoter. Such peculiar synergistic transcription activation has not yet been observed in other aromatic catabolic pathways, and the MhpR activator becomes the first member of the IclR family of transcriptional regulators that is indispensable for recruiting CRP to the target promoter.

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disorder of still unknown etiology that results in loss of motoneurons, paralysis, and death, usually between 2 and 4 years from onset. There are no currently available ALS biomarkers to support early diagnosis and to facilitate the assessment of the efficacy of new treatments. Since ALS is considered a multisystemic disease, here we have investigated the usefulness of immortalized lymphocytes from sporadic ALS patients to study TDP-43 homeostasis as well as to provide a convenient platform to evaluate TDP-43 phosphorylation as a novel therapeutic approach for ALS. We report here that lymphoblasts from ALS patients recapitulate the hallmarks of TDP-43 processing in affected motoneurons, such as increased phosphorylation, truncation, and mislocalization of TDP-43. Moreover, modulation of TDP-43 by an in-house designed protein casein kinase-1δ (CK-1δ) inhibitor, IGS3.27, reduced phosphorylation of TDP-43, and normalized the nucleo-cytosol translocation of TDP-43 in ALS lymphoblasts. Therefore, we conclude that lymphoblasts, easily accessible cells, from ALS patients could be a useful model to study pathological features of ALS disease and a suitable platform to test the effects of potential disease-modifying drugs even in a personalized manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.

Podocalyxin (Podxl) has an essential role in the development and function of the kidney glomerular filtration barrier. It is also expressed by vascular endothelia but perinatal lethality of podxl(-/-) mice has precluded understanding of its function in adult vascular endothelial cells (ECs). In this work, we show that conditional knockout mice with deletion of Podxl restricted to the vascular endothelium grow normally but most die spontaneously around three months of age. Histological analysis showed a nonspecific inflammatory infiltrate within the vessel wall frequently associated with degenerative changes, and involving vessels of different caliber in one or more organs. Podxl-deficient lung EC cultures exhibit increased permeability to dextran and macrophage transmigration. After thrombin stimulation, ECs lacking Podxl showed delayed recovery of VE-cadherin cell contacts, persistence of F-actin stress fibers, and sustained phosphorylation of the ERM complex and activation of RhoA, suggesting a failure in endothelial barrier stabilization. The results suggest that Podxl has an essential role in the regulation of endothelial permeability by influencing the mechanisms involved in the restoration of endothelial barrier integrity after injury.