Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Public Art, strategies for the regeneration of public space

This paper discusses some of the key concepts in the consideration of public art as a central element in urban regeneration processes, especially in reference to its role in the processes of citizen participation.

Gradients and regeneration: the case of TNEX59 in planarians (Platyhelminthes, Tricladida)

During development, multicellular organisms determine and then differentiate regions that constitute the body's architecture. These regions are established and controlled by a number of molecules, including nuclear factors, that drive the organism from the egg to its final shape. We studied the molecules involved in the regionalisation of the freshwater planarian body (Platyhelminthes, Turbelleria, Tricladida).

Orofacial pain of cardiac origin: review literature and clinical cases

The most common types of orofacial pain originate at the dental or periodontal level or in the musculoskeletal structures. However, the patient may present pain in this region even though the source is located elsewhere in the body. One possible source of heterotopic pain is of cardiac origin. Objectives: Report two cases of orofacial pain of cardiac origin and review the clinical cases described in the literature. Study Design: Description of clinical cases and review of clinical cases. Results and conclusions: Nine cases of atypical pain of cardiac origin are recorded, which include 5 females and 4 males. In craniofacial structures, pain of cardiac origin is usually bilateral. At the craniofacial level, the most frequent location described is in the throat and jaw. Pain of cardiac origin is considered atypical due to its location, although roughly 10% of the cases of cardiac ischemia manifest primarily in craniofacial structures. Finally, the differential diagnosis of pain of odontogenic origin must be taken into account with pain of non-odontogenic origin (muscle, psychogenic, neuronal, cardiac, sinus and neurovascular pain) in order to avoid diagnostic errors in the dental practice as well as unnecessary treatments.

Persistence of tracer in the application site -a potential confounding factor in nerve regeneration studies

Selective reinnervation of peripheral targets after nerve injury might be assessed by injecting a first tracer in a target before nerve injury to label the original neuronal population, and applying a second tracer after the regeneration period to label the regenerated population. However, altered uptake of tracer, fading, and cell death may interfere with the results. Furthermore, if the first tracer injected remains in the target tissue, available for 're-uptake' by misdirected regenerating axons, which originally innervated another region, then the identification of the original population would be confused. With the aim of studying this problem, the sciatic nerve of adult rats was sectioned and sutured. After 3 days, to allow the distal axon to degenerate avoiding immediate retrograde transport, one of the dyes: Fast Blue (FB), Fluoro-Gold (FG) or Diamidino Yellow (DY), was injected into the tibial branch of the sciatic nerve, or in the skin of one of the denervated digits. Rats survived 2-3 months. The results showed labelled dorsal root ganglion (DRG) cells and motoneurones, indicating that late re-uptake of a first tracer occurs. This phenomenon must be considered when the model of sequential labelling is used for studying the accuracy of peripheral reinnervation.

Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration

Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results have high potential to generate significant information for development of regenerative and anti cancer therapies.

The Interplay between NF-kappaB and E2F1 Coordinately Regulates Inflammation and Metabolism in Human Cardiac Cells

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.

Orofacial pain of cardiac origin, serial of clinical cases

Objective: To determine the clinical characteristics of the orofacial pain of cardiac origin in patients visited when doing a treadmill exercise test, at the cardiology service of the Can Ruti Hospital in Badalona (Barcelona, Spain). Study design: The sample of that study included thirty patients visiteding when doing a treadmill exercise test, at the cardiology service. The questionnaire has been asked to a sample of 30 patients. Results: Eleven of the 30 patients included in this study presented craniofacial pain before or during the cardiac seizure. The location of the pain was bilateral, non-irradiated at the mandible in all cases. The intensity of the pain was from slight to severe. The frequency of the appearance of the pain was paroxysmal in 8 cases and constant in three cases, and the duration was from a few hours to a maximum of 14 days. Discussion: The cardiac pain in craniofacial structures is usually bilateral, compared to odontogenic pain which is always unilateral. The pain of cardiac origin is considered atypical because of its location, but about the 10 % of the cases, the cardiac ischemia has its primary manifestation in orofacial structures. Conclusions: Eleven patients referred a bilateral non-irradiated mandibular pain, with intensity from slight to severe, and with a paroxystic frequency in eight cases and a constant frequency in three cases. Just one patient referred pain during the treadmill exercise test. In all cases the pain disappeared after the cardiac surgery or the administration of vasodilators.

Estimations of topographically correct regeneration to nerve branches and skin after peripheral nerve injury and repair.

Peripheral nerve injury is typically associated with long-term disturbances in sensory localization, despite nerve repair and regeneration. Here, we investigate the extent of correct reinnervation by back-labeling neuronal soma with fluorescent tracers applied in the target area before and after sciatic nerve injury and repair in the rat. The subpopulations of sensory or motor neurons that had regenerated their axons to either the tibial branch or the skin of the third hindlimb digit were calculated from the number of cell bodies labeled by the first and/or second tracer. Compared to the normal control side, 81% of the sensory and 66% of the motor tibial nerve cells regenerated their axons back to this nerve, while 22% of the afferent cells from the third digit reinnervated this digit. Corresponding percentages based on quantification of the surviving population on the experimental side showed 91%, 87%, and 56%, respectively. The results show that nerve injury followed by nerve repair by epineurial suture results in a high but variable amount of topographically correct regeneration, and that proportionally more neurons regenerate into the correct proximal nerve branch than into the correct innervation territory in the skin

JNK controls the onset of mitosis of planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun-NH2-kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.

Cardiac tissue engineering by electrical stimulation with subcutaneous and cardiac adipose-tissue derived progenitor cells (ATDPCs)

A major challenge of cardiac tissue engineering is directing cells to establish the physiological structure and function of the myocardium being replaced. In native heart, pacing cells generate electrical stimuli that spread throughout the heartcausing cell membrane depolarization and activation of contractile apparatus. We ought to examine whether electricalstimulation of adipose tissue-derived progenitor cells (ATDPCs) exerts phenotypic and genetic changes that enhance theircardiomyogenic potential.

Cost-effectiveness analysis of cardiac resynchronization therapy in patients with NYHA I and NYHA II heart failure in Spain

Objectives: The aim of the study was to combine clinical results from the European Cohort of the REVERSE study and costs associated with the addition of cardiac resynchronization therapy (CRT) to optimal medical therapy (OMT) in patients with mild symptomatic (NYHA I-II) or asymptomatic left ventricular dysfunction and markers of cardiac dyssynchrony in Spain. Methods: A Markov model was developed with CRT + OMT (CRT-ON) versus OMT only (CRT-OFF) based on a retrospective cost-effectiveness analysis. Raw data was derived from literature and expert opinion, reflecting clinical and economic consequences of patient"s management in Spain. Time horizon was 10 years. Both costs (euro 2010) and effects were discounted at 3 percent per annum. Results: CRT-ON showed higher total costs than CRT-OFF; however, CRT reduced the length of hospitalization in ICU by 94 percent (0.006 versus 0.091 days) and general ward in by 34 percent (0.705 versus 1.076 days). Surviving CRT-ON patients (88.2 percent versus 77.5 percent) remained in better functional class longer, and they achieved an improvement of 0.9 life years (LYGs) and 0.77 years quality-adjusted life years (QALYs). CRT-ON proved to be cost-effective after 6 years, except for the 7th year due to battery depletion. At 10 years, the results were 18,431 per LYG and 21,500 per QALY gained. Probabilistic sensitivity analysis showed CRT-ON was cost-effective in 75.4 percent of the cases at 10 years. Conclusions: The use of CRT added to OMT represents an efficient use of resources in patients suffering from heart failure in NYHA functional classes I and II.

Association of early repolarization with risk of cardiac mortality in the general population

Early repolarization, which is characterized by an elevation of the J-point on 12-lead electrocardiography, is a common finding that has been considered as benign for decades. However, in the last years, it has been related with vulnerability to idiopathic ventricular fibrillation and with cardiac mortality in the general population. Recently, 4 potential ECG predictors that could differentiate the benign from the malignant form of early repolarization have been suggested. Any previous study about early repolarization has been done in Spain. Aim. To ascertain whether the presence of early repolarization pattern in a resting electrocardiogram is associated with a major risk of cardiac death in a Spanish general population and to determine whether the presence of potential predictors of malignancy in a resting electrocardiogram increases the risk of cardiac mortality in patients with early repolarization pattern. Methods. We will analyse the presence of early repolarization and the occurrence of cardiac mortality in a retrospective cohort study of 4,279 participants aged 25 to 74 years in the province of Girona. This cohort has been followed during a mean of 9.8 years. Early repolarization will be stratified according to the degree of J-point elevation (≥0.1 mV or ≥0.2 mV), the morphology of the J-wave (slurring, notching or any of these two), the ST-segment pattern (ascending or descending) and the localization (inferior leads, lateral leads, or both). Association of early repolarization with cardiac death will be assessed by adjusted Cox-proportional hazards models

A Novel Missense Mutation, I890T, in the Pore Region of Cardiac Sodium Channel Causes Brugada Syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na₊ channel alpha subunit (Naᵥ1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Wild-type (WT) or I890T Naᵥ1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Naᵥ1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from -52.0 ± 6.5 pA/pF, n=15 to 35.9 ± 3.4 pA/pF, n = 22, at -20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V½ =-32.0 ± 0.3 mV, n = 18, and -27.3 ± 0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Nav1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Nav1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation