Macrophages modulate fibrosis during newt lens regeneration.

Background: Previous studies indicated that macrophages play a role during lens regeneration in newts, but their function has not been tested experimentally. Methods: Here we generated a transgenic newt reporter line in which macrophages can be visualized in vivo. Using this new tool, we analyzed the location of macrophages during lens regeneration. We uncovered early gene expression changes using bulk RNAseq in two newt species, Notophthalmus viridescens and Pleurodeles waltl. Next, we used clodronate liposomes to deplete macrophages, which inhibited lens regeneration in both newt species. Results: Macrophage depletion induced the formation of scar-like tissue, an increased and sustained inflammatory response, an early decrease in iris pigment epithelial cell (iPEC) proliferation and a late increase in apoptosis. Some of these phenotypes persisted for at least 100 days and could be rescued by exogenous FGF2. Re-injury alleviated the effects of macrophage depletion and re-started the regeneration process. Conclusions: Together, our findings highlight the importance of macrophages in facilitating a pro-regenerative environment in the newt eye, helping to resolve fibrosis, modulating the overall inflammatory landscape and maintaining the proper balance of early proliferation and late apoptosis.

Understanding the microenvironment and how this controls cell fate.

The microenvironment influences cell fate. In this collection of voices, researchers from the fields of cancer and regeneration highlight approaches to establish the importance of the microenvironment and discuss future directions to understand the complex interaction between cells and their surrounding environment and how this impacts on disease and regeneration.

Useful Electrocardiographic Signs to Support the Prediction of Favorable Response to Cardiac Resynchronization Therapy.

Cardiac resynchronization therapy (CRT) is a cornerstone therapeutic opportunity for selected patients with heart failure. For optimal patient selection, no other method has been proven to be more effective than the 12-lead ECG, and hence ECG characteristics are extensively researched. The evaluation of particular ECG signs before the implantation may improve selection and, consequently, clinical outcomes. The definition of a true left bundle branch block (LBBB) seems to be the best starting point with which to select patients for CRT. Although there are no universally accepted definitions of LBBB, using the classical LBBB criteria, some ECG parameters are associated with CRT response. In patients with non-true LBBB or non-LBBB, further ECG predictors of response and non-response could be analyzed, such as QRS fractionation, signs of residual left bundle branch conduction, S-waves in V6, intrinsicoid deflection, or non-invasive estimates of Q-LV which are described in newer publications. The most important and recent study results of the topic are summarized and discussed in this current review.

Synthesis and Anticancer Activity of Phosphinoylated and Phosphonoylated N-Heterocycles Obtained by the Microwave-Assisted Palladium Acetate-Catalyzed Hirao Reaction.

A literature survey showed that different derivatives with the 9-phenyl-9H-carbazole or the dihydroindoline scaffold may be of biological activity including cytotoxic effect. Driven by this experience, P-functionalized derivatives of these N-heterocycles were synthesized. Three N-heterocycles, 9-(4-bromophenyl)-9H-carbazole, 3-bromo-9-phenyl-9H-carbazole and 1-(5-bromoindolin-1-yl)ethan-1-one, were coupled with dialkyl phosphites and diarylphosphine oxides using Pd(OAc)2 (10 %) as the catalyst precursor and triethylamine as the base in ethanol under microwave irradiation. The excess of the Y2 P(O)H reagent (Y=alkoxy, aryl) (30 %) served as the P-ligand in its trivalent tautomeric form (Y2 POH), hence there was no need for the usual P-ligands meaning cost and environmental burden. Hence, the presented method is a "green" approach that proved to be more efficient than the preparation by the traditional method. The products, dialkyl phosphonates and tertiary phosphine oxides obtained in 58-84 % yields were characterized, one of them also by single crystal X-ray analysis, and were subjected to in vitro biological activity evaluation. A (carbazol)yl-phenylphosphonate, an N-phenyl-(carbazol)yl-phosphonate, a (carbazol)yl-phenylphosphine oxide and an N-phenyl-(carbazol)ylphosphine oxide revealed a significant cytotoxic activity on A549 human non-small-cell lung carcinoma and MonoMac-6 acute monocytic leukemia cancer cells. The cytotoxic effect was significant as compared to that of the reference compounds.

A proposal for better visualization of Aslanger pattern.

In the context of reporting a case of a patient with Aslanger pattern, the application of additional leads in the frontal plane is proposed to facilitate the recognition of this condition - indicating occlusive myocardial infarction - as eligible for the current ST-segment elevation myocardial infarction (STEMI) criteria and consequently for immediate reperfusion strategy recommendation.

Macrophages modulate fibrosis during newt lens regeneration.

Previous studies indicated that macrophages play a role during lens regeneration in newts, but their function has not been tested experimentally. Here we generated a transgenic newt reporter line in which macrophages can be visualized in vivo. Using this new tool, we analyzed the location of macrophages during lens regeneration. We uncovered early gene expression changes using bulk RNAseq in two newt species, Notophthalmus viridescens and Pleurodeles waltl. Next, we used clodronate liposomes to deplete macrophages, which inhibited lens regeneration in both newt species. Macrophage depletion induced the formation of scar-like tissue, an increased and sustained inflammatory response, an early decrease in iris pigment epithelial cell (iPEC) proliferation and a late increase in apoptosis. Some of these phenotypes persisted for at least 100 days and could be rescued by exogenous FGF2. Re-injury alleviated the effects of macrophage depletion and re-started the regeneration process. Together, our findings highlight the importance of macrophages in facilitating a pro-regenerative environment in the newt eye, helping to resolve fibrosis, modulating the overall inflammatory landscape and maintaining the proper balance of early proliferation and late apoptosis.

Evaluation of genetic demultiplexing of single-cell sequencing data from model species.

Single-cell sequencing (sc-seq) provides a species agnostic tool to study cellular processes. However, these technologies are expensive and require sufficient cell quantities and biological replicates to avoid artifactual results. An option to address these problems is pooling cells from multiple individuals into one sc-seq library. In humans, genotype-based computational separation (i.e., demultiplexing) of pooled sc-seq samples is common. This approach would be instrumental for studying non-isogenic model organisms. We set out to determine whether genotype-based demultiplexing could be more broadly applied among species ranging from zebrafish to non-human primates. Using such non-isogenic species, we benchmark genotype-based demultiplexing of pooled sc-seq datasets against various ground truths. We demonstrate that genotype-based demultiplexing of pooled sc-seq samples can be used with confidence in several non-isogenic model organisms and uncover limitations of this method. Importantly, the only genomic resource required for this approach is sc-seq data and a de novo transcriptome. The incorporation of pooling into sc-seq study designs will decrease cost while simultaneously increasing the reproducibility and experimental options in non-isogenic model organisms.

The Application of a New, Modified Algorithm for the Differentiation of Regular Ventricular and Pre-Excited Tachycardias.

BACKGROUND: The three-step Brugada group algorithm is the only published electrocardiographical (ECG) algorithm for differentiating ventricular tachycardia (VT) from pre-excited tachycardia (PXT) as a cause of regular wide QRS complex tachycardia (WCT). This study aimed to improve the diagnostic accuracy of the Brugada group algorithm. METHODS: This study modified the Brugada group algorithm by adding a new aVR lead criterion (initial positive deflection in lead aVR and the QRS complex area above the baseline is greater than the area below the baseline). The Brugada group algorithm and the new, modified four-step algorithm in 300 WCT ECGs (241 VTs, 59 PXTs) was applied. If any of the criteria were fulfilled, VT was diagnosed; if none were fulfilled, a diagnosis of PXT was established. RESULTS: The test accuracy, VT diagnosis sensitivity, and negative predictive value (NPV) of the new, modified algorithm were significantly greater than that of the Brugada group algorithm: test accuracy 220 of 300 (73%) vs 182 of 300 (61%); sensitivity 73% vs 55% (p<0.001 for both); NPV 40% vs 31% (p=0.0205). The VT diagnosis specificity of the Brugada group algorithm was greater than that of the new, modified algorithm (83% vs 75%; p=0.019). There was no significant difference between the new, modified and Brugada group algorithms in the positive predictive values (92% vs 93%, respectively) for a VT diagnosis, and positive and negative likelihood ratio values (2.87 vs 3.26; 0.36 vs 0.54, respectively). CONCLUSIONS: The new, modified algorithm proved to be more sensitive for the differentiation of VT from PXT than the Brugada group algorithm.

A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.

Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.

New N-acyl- as well as N-phosphonoylmethyl- and N-phosphinoylmethyl-α-amino-benzylphosphonates by acylation and a tandem Kabachnik-Fields protocol.

Diethyl α-benzylamino- and α-amino-benzylphosphonates obtained by the Kabachnik-Fields reaction were useful intermediates in the synthesis of other derivatives. Acylation of α-aminophosphonates with acyl chlorides led to the corresponding N-acyl species existing under a dynamic equilibrium of two conformers. Judging from the broad NMR signals, the sterically most crowded N-benzoyl-N-benzyl derivative suffered a hindered rotation around the N-C axis to the acyl carbon atom at 26 °C. Low temperature NMR measurements at -10 °C showed the presence of two distinct rotamers that were characterized. The other acylated α-amino-benzylphosphonates prepared revealed a less hindered rotation. Single crystal X-ray diffraction of the NH-propionyl species showed a dimer, in which the two molecules were held together by rare intermolecular PO⋯HN bonds. On the other hand, substituted α-benzylamino-benzylphosphonates prepared by phospha-Mannich reactions were employed, as a new approach, in a second Kabachnik-Fields condensation by reaction with formaldehyde and dialkyl phosphites or secondary phosphine oxides to afford novel N-phosphonoylmethyl- and N-phosphinoylmethyl-α-amino-benzylphosphonates. The structure of the new products was confirmed by two-dimensional NMR spectroscopy. A symmetrical bis derivative was prepared in a diastereoselective manner. A related tris(phosphonoylmethyl)amine species was also synthesized.

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration.

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

Cell-type profiling in salamanders identifies innovations in vertebrate forebrain evolution.

The evolution of advanced cognition in vertebrates is associated with two independent innovations in the forebrain: the six-layered neocortex in mammals and the dorsal ventricular ridge (DVR) in sauropsids (reptiles and birds). How these innovations arose in vertebrate ancestors remains unclear. To reconstruct forebrain evolution in tetrapods, we built a cell-type atlas of the telencephalon of the salamander Pleurodeles waltl. Our molecular, developmental, and connectivity data indicate that parts of the sauropsid DVR trace back to tetrapod ancestors. By contrast, the salamander dorsal pallium is devoid of cellular and molecular characteristics of the mammalian neocortex yet shares similarities with the entorhinal cortex and subiculum. Our findings chart the series of innovations that resulted in the emergence of the mammalian six-layered neocortex and the sauropsid DVR.

Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders.

The contribution of the epicardium, the outermost layer of the heart, to cardiac regeneration has remained controversial due to a lack of suitable analytical tools. By combining genetic marker-independent lineage-tracing strategies with transcriptional profiling and loss-of-function methods, we report here that the epicardium of the highly regenerative salamander species Pleurodeles waltl has an intrinsic capacity to differentiate into cardiomyocytes. Following cryoinjury, CLDN6+ epicardium-derived cells appear at the lesion site, organize into honeycomb-like structures connected via focal tight junctions and undergo transcriptional reprogramming that results in concomitant differentiation into de novo cardiomyocytes. Ablation of CLDN6+ differentiation intermediates as well as disruption of their tight junctions impairs cardiac regeneration. Salamanders constitute the evolutionarily closest species to mammals with an extensive ability to regenerate heart muscle and our results highlight the epicardium and tight junctions as key targets in efforts to promote cardiac regeneration.

Tig1 regulates proximo-distal identity during salamander limb regeneration.

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Synthesis of Novel Crown Ether-Squaramides and Their Application as Phase-Transfer Catalysts.

This work presents the synthesis of six new phase-transfer organocatalysts in which the squaramide unit is directly linked to the nitrogen atom of an aza-crown ether. Four chiral skeletons, namely hydroquinine, quinine, cinchonine (cinchonas), and α-d-glucopyranoside were responsible for the asymmetric construction of an all-carbon quaternary stereogenic center in α-alkylation and Michael addition reactions of malonic esters. We investigated the effects of these different chiral units and that of crown ethers with different sizes on catalytic activity and enantioselectivity. During extensive parameter investigations, both conventional and emerging green solvents were screened, providing valuable α,α-disubstituted malonic ester derivatives with excellent yields (up to 98%).

Locomotion dependent neuron-glia interactions control neurogenesis and regeneration in the adult zebrafish spinal cord.

Physical exercise stimulates adult neurogenesis, yet the underlying mechanisms remain poorly understood. A fundamental component of the innate neuroregenerative capacity of zebrafish is the proliferative and neurogenic ability of the neural stem/progenitor cells. Here, we show that in the intact spinal cord, this plasticity response can be activated by physical exercise by demonstrating that the cholinergic neurotransmission from spinal locomotor neurons activates spinal neural stem/progenitor cells, leading to neurogenesis in the adult zebrafish. We also show that GABA acts in a non-synaptic fashion to maintain neural stem/progenitor cell quiescence in the spinal cord and that training-induced activation of neurogenesis requires a reduction of GABAA receptors. Furthermore, both pharmacological stimulation of cholinergic receptors, as well as interference with GABAergic signaling, promote functional recovery after spinal cord injury. Our findings provide a model for locomotor networks' activity-dependent neurogenesis during homeostasis and regeneration in the adult zebrafish spinal cord.

Nerve-associated Schwann cell precursors contribute extracutaneous melanocytes to the heart, inner ear, supraorbital locations and brain meninges.

Melanocytes are pigmented cells residing mostly in the skin and hair follicles of vertebrates, where they contribute to colouration and protection against UV-B radiation. However, the spectrum of their functions reaches far beyond that. For instance, these pigment-producing cells are found inside the inner ear, where they contribute to the hearing function, and in the heart, where they are involved in the electrical conductivity and support the stiffness of cardiac valves. The embryonic origin of such extracutaneous melanocytes is not clear. We took advantage of lineage-tracing experiments combined with 3D visualizations and gene knockout strategies to address this long-standing question. We revealed that Schwann cell precursors are recruited from the local innervation during embryonic development and give rise to extracutaneous melanocytes in the heart, brain meninges, inner ear, and other locations. In embryos with a knockout of the EdnrB receptor, a condition imitating Waardenburg syndrome, we observed only nerve-associated melanoblasts, which failed to detach from the nerves and to enter the inner ear. Finally, we looked into the evolutionary aspects of extracutaneous melanocytes and found that pigment cells are associated mainly with nerves and blood vessels in amphibians and fish. This new knowledge of the nerve-dependent origin of extracutaneous pigment cells might be directly relevant to the formation of extracutaneous melanoma in humans.

Effect of N,N'-Dicyclohexyldicarboxamide Homologues on the Crystallization and Properties of Isotactic Polypropylene.

Application of nucleating agents is the most versatile and industrially applied way to manipulate the crystalline structure of isotactic polypropylene (iPP). Various materials possess a nucleating effect, but from the viewpoint of dispersibility, the partially soluble ones are the most advantageous. Our objective was to synthesize new N,N'-dicyclohexyldicarboxamide homologues and study their applicability as nucleating agents in iPP. Carbon-13 nuclear magnetic resonance (13C NMR) and infrared spectroscopy were used to prove that the synthesis reactions were successful. Thermal stability of the compounds was investigated with simultaneous thermal analysis. Nucleating efficiency and solubility were characterized by polarized light microscopy and differential scanning calorimetry. Polarized light microscopy was also applied to study the effect of novel additives on the morphology of iPP. The properties, important from the viewpoint of applicability, were also investigated. Tensile tests were performed to characterize the main mechanical properties, and standard haze measurements were performed to characterize optical properties. It can be concluded that the investigated compounds are partially soluble nucleating agents and influence the crystalline structure of iPP. Most of the studied compounds have a moderate nucleating efficiency, but a very interesting dendritic structure develops in their presence. Two of them proved to be non-selective ß-nucleating agents, which result in a remarkable improvement of impact resistance and higher opacity.