Reassessment of miRNA variant (isomiRs) composition by small RNA sequencing.

IsomiRs, sequence variants of mature microRNAs, are usually detected and quantified using high-throughput sequencing. Many examples of their biological relevance have been reported, but sequencing artifacts identified as artificial variants might bias biological inference and therefore need to be ideally avoided. We conducted a comprehensive evaluation of 10 different small RNA sequencing protocols, exploring both a theoretically isomiR-free pool of synthetic miRNAs and HEK293T cells. We calculated that, with the exception of two protocols, less than 5% of miRNA reads can be attributed to library preparation artifacts. Randomized-end adapter protocols showed superior accuracy, with 40% of true biological isomiRs. Nevertheless, we demonstrate concordance across protocols for selected miRNAs in non-templated uridyl additions. Notably, NTA-U calling and isomiR target prediction can be inaccurate when using protocols with poor single-nucleotide resolution. Our results highlight the relevance of protocol choice for biological isomiRs detection and annotation, which has key potential implications for biomedical applications.

Targeting metabolic plasticity in glioma stem cells in vitro and in vivo through specific inhibition of c-Src by TAT-Cx43266-283.

BACKGROUND: Glioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43266-283 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43266-283. METHODS: Metabolic impairment induced by the c-Src inhibitor TAT-Cx43266-283 in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence. FINDINGS: TAT-Cx43266-283 decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43266-283 impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43266-283 showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3. INTERPRETATION: The reduced ability of TAT-Cx43266-283-treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43266-283. FUNDING: Spanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).

Connexin43 Region 266-283, via Src Inhibition, Reduces Neural Progenitor Cell Proliferation Promoted by EGF and FGF-2 and Increases Astrocytic Differentiation.

Neural progenitor cells (NPCs) are self-renewing cells that give rise to the major cells in the nervous system and are considered to be the possible cell of origin of glioblastoma. The gap junction protein connexin43 (Cx43) is expressed by NPCs, exerting channel-dependent and -independent roles. We focused on one property of Cx43-its ability to inhibit Src, a key protein in brain development and oncogenesis. Because Src inhibition is carried out by the sequence 266-283 of the intracellular C terminus in Cx43, we used a cell-penetrating peptide containing this sequence, TAT-Cx43266-283, to explore its effects on postnatal subventricular zone NPCs. Our results show that TAT-Cx43266-283 inhibited Src activity and reduced NPC proliferation and survival promoted by epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). In differentiation conditions, TAT-Cx43266-283 increased astrocyte differentiation at the expense of neuronal differentiation, which coincided with a reduction in Src activity and ß-catenin expression. We propose that Cx43, through the region 266-283, reduces Src activity, leading to disruption of EGF and FGF-2 signaling and to down-regulation of ß-catenin with effects on proliferation and differentiation. Our data indicate that the inhibition of Src might contribute to the complex role of Cx43 in NPCs and open new opportunities for further research in gliomagenesis.

Safety Assessment of Benzyne Generation from a Silyl Triflate Precursor.

Silyl triflate precursors to benzyne and related intermediates have emerged as valuable synthetic building blocks. However, data addressing the safety of employing these silyl triflate precursors are lacking. We report the calorimetric analysis of a typical Kobayashi procedure for forming and trapping benzyne using a silyl triflate precursor. Our findings suggest that, unlike benzenediazonium carboxylate precursors to benzyne, silyl triflates may be employed under mild conditions without severe concern for runaway reaction.

Connexin43 peptide, TAT-Cx43266-283, selectively targets glioma cells, impairs malignant growth, and enhances survival in mouse models in vivo.

BACKGROUND: Malignant gliomas are the most frequent primary brain tumors and remain among the most incurable cancers. Although the role of the gap junction protein, connexin43 (Cx43), has been deeply investigated in malignant gliomas, no compounds have been reported with the ability to recapitulate the tumor suppressor properties of this protein in in vivo glioma models. METHODS: TAT-Cx43266-283 a cell-penetrating peptide which mimics the effect of Cx43 on c-Src inhibition, was studied in orthotopic immunocompetent and immunosuppressed models of glioma. The effects of this peptide in brain cells were also analyzed. RESULTS: While glioma stem cell malignant features were strongly affected by TAT-Cx43266-283, these properties were not significantly modified in neurons and astrocytes. Intraperitoneally administered TAT-Cx43266-283 decreased the invasion of intracranial tumors generated by GL261 mouse glioma cells in immunocompetent mice. When human glioma stem cells were intracranially injected with TAT-Cx43266-283 into immunodeficient mice, there was reduced expression of the stemness markers nestin and Sox2 in human glioma cells at 7 days post-implantation. Consistent with the role of Sox2 as a transcription factor required for tumorigenicity, TAT-Cx43266-283 reduced the number and stemness of human glioma cells at 30 days post-implantation. Furthermore, TAT-Cx43266-283 enhanced the survival of immunocompetent mice bearing gliomas derived from murine glioma stem cells. CONCLUSION: TAT-Cx43266-283 reduces the growth, invasion, and progression of malignant gliomas and enhances the survival of glioma-bearing mice without exerting toxicity in endogenous brain cells, which suggests that this peptide could be considered as a new clinical therapy for high-grade gliomas.

Cu-Catalyzed Hydroboration of Benzylidenecyclopropanes: Reaction Optimization, (Hetero)Aryl Scope, and Origins of Pathway Selectivity.

The copper-catalyzed hydroboration of benzylidenecyclopropanes, conveniently accessed in one step from readily available benzaldehydes, is reported. Under otherwise identical reaction conditions, two distinct phosphine ligands grant access to different products by either suppressing or promoting cyclopropane opening via ß-carbon elimination. Computational studies provide insight into how the rigidity and steric environment of these different bis-phosphine ligands influence the relative activation energies of ß-carbon elimination versus protodecupration from the key benzylcopper intermediate. The method tolerates a wide variety of heterocycles prevalent in clinical and pre-clinical drug development, giving access to valuable synthetic intermediates. The versatility of the tertiary cyclopropylboronic ester products is demonstrated through several derivatization reactions.

Endocytosis and Transcytosis of Amyloid-ß Peptides by Astrocytes: A Possible Mechanism for Amyloid-ß Clearance in Alzheimer's Disease.

Amyloid-ß (Aß) peptides, Aß40, Aß42, and recently Aß25 - 35, have been directly implicated in the pathogenesis of Alzheimer's disease (AD). We have previously shown that all three peptides decrease neuronal viability, but Aß40 also promotes synaptic disassembling. In this work, we have studied the effects of these peptides on astrocytes in primary culture and found that the three Aß peptides were internalized by astrocytes and significantly decreased astrocyte viability, while increasing ROS production. Aß peptide internalization is temperature-dependent, a fact that supports the idea that Aß peptides are actively endocytosed by astrocytes. However, inhibiting caveolae formation by methyl-beta-cyclodextrin or by silencing caveolin-1 with RNA interference did not prevent Aß endocytosis, which suggests that Aß peptides do not use caveolae to enter astrocytes. Conversely, inhibition of clathrin-coated vesicle formation by chlorpromazine or by silencing clathrin with RNA interference significantly decreased Aß internalization and partially reverted the decrease of astrocyte viability caused by the presence of Aß. These results suggest that Aß is endocytosed by clathrin-coated vesicles in astrocytes. Aß-loaded astrocytes, when co-incubated with non-treated astrocytes in separate wells but with the same incubation medium, promoted cell death in non-treated astrocytes; a fact that was associated with the presence of Aß inside previously unloaded astrocytes. This phenomenon was inhibited by the presence of chlorpromazine in the co-incubation medium. These results suggest that astrocyte may perform Aß transcytosis, a process that could play a role in the clearance of Aß peptides from the brain to cerebrospinal fluid.

Direct Access to Versatile Electrophiles via Catalytic Oxidative Cyanation of Alkenes.

Nucleophilic attack on carbon-based electrophiles is a central reactivity paradigm in chemistry and biology. The steric and electronic properties of the electrophile dictate its reactivity with different nucleophiles of interest, allowing the opportunity to fine-tune electrophiles for use as coupling partners in multistep organic synthesis or for covalent modification of proteins in drug discovery. Reactions that directly transform inexpensive chemical feedstocks into versatile carbon electrophiles would therefore be highly enabling. Herein, we report the catalytic, regioselective oxidative cyanation of conjugated and nonconjugated alkenes using a homogeneous copper catalyst and a bystanding N-F oxidant to furnish branched alkenyl nitriles that are difficult to prepare using existing methods. We show that the alkenyl nitrile products serve as electrophilic reaction partners for both organic synthesis and the chemical proteomic discovery of covalent protein ligands.

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

Expanding the ROMP Toolbox: Synthesis of Air-Stable Benzonorbornadiene Polymers by Aryne Chemistry.

Benzonorbornadiene polymers synthesized by ring-opening metathesis polymerization (ROMP) are typically prone to oxidation at the benzylic/allylic position under ambient conditions. Accordingly, the use of benzonorbornadiene polymers in practical applications has remained limited. In this manuscript, we report the synthesis of poly(benzonorbornadiene) polymers using a strategic blend of benzyne chemistry and ROMP. Through a comparative study, we show that substitution at the benzylic/allylic position prevents oxidative deformation, yet does not inhibit polymerization by common ruthenium catalysts with good control over molecular weight dispersity. We expect the benzyne/ROMP reaction sequence will allow easy access to air-stable benzonorbornadiene polymers for various applications.

A Short Region of Connexin43 Reduces Human Glioma Stem Cell Migration, Invasion, and Survival through Src, PTEN, and FAK.

Connexin43 (CX43), a protein that forms gap junction channels and hemichannels in astrocytes, is downregulated in high-grade gliomas. Its relevance for glioma therapy has been thoroughly explored; however, its positive effects on proliferation are counterbalanced by its effects on migration and invasion. Here, we show that a cell-penetrating peptide based on CX43 (TAT-Cx43266-283) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensin homolog in glioma stem cells (GSCs) derived from patients. Consequently, TAT-Cx43266-283 reduced GSC motility, as analyzed by time-lapse microscopy, and strongly reduced their invasive ability. Interestingly, we investigated the effects of TAT-Cx43266-283 on freshly removed surgical specimens as undissociated glioblastoma blocks, which revealed a dramatic reduction in the growth, migration, and survival of these cells. In conclusion, a region of CX43 (amino acids 266-283) exerts an important anti-tumor effect in patient-derived glioblastoma models that includes impairment of GSC migration and invasion.

Mizoroki-Heck Cyclizations of Amide Derivatives for the Introduction of Quaternary Centers.

We report non-decarbonylative Mizoroki-Heck reactions of amide derivatives. The transformation relies on the use of nickel catalysis and proceeds using sterically hindered tri- and tetrasubstituted olefins to give products containing quaternary centers. The resulting polycyclic or spirocyclic products can be obtained in good yields. Moreover, a diastereoselective variant of this method gives access to an adduct bearing vicinal, highly substituted sp3 stereocenters. These results demonstrate that amide derivatives can be used as building blocks for the assembly of complex scaffolds.

Restrained Phosphatidylcholine Synthesis in a Cellular Model of Down's Syndrome is Associated with the Overexpression of Dyrk1A.

Aberrant formation of the cerebral cortex could be attributed to the lack of suitable substrates that direct the migration of neurons. Previous work carried out at our laboratory has shown that oleic acid is a neurotrophic factor. In order to characterize the effect of oleic acid in a cellular model of Down's syndrome (DS), here, we used immortalized cell lines derived from the cortex of trisomy Ts16 and euploid mice. We report that in the plasma membrane of euploid cells, an increase in phosphatidylcholine concentrations occurs in the presence of oleic acid. However, in trisomic cells, oleic acid failed to increase phosphatidylcholine incorporation into the plasma membrane. Gene expression analysis of trisomic cells revealed that the phosphatidylcholine biosynthetic pathway was deregulated. Taken together, these results suggest that the overdose of specific genes in trisomic lines delays differentiation in the presence of oleic acid. The dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human chromosome 21. DYRK1A contributes to intellectual disability and the early onset of Alzheimer's disease in DS patients. Here, we explored the potential role of Dyrk1A in the reduction of phosphatidylcholine concentrations in trisomic cells in the presence of oleic acid. The downregulation of Dyrk1A by small interfering RNA (siRNA) in trisomic cells returned phosphatidylcholine concentrations up to similar levels to those of euploid cells in the presence of oleic acid. Thus, our results highlight the role of Dyrk1A in brain development through the modulation of phosphatidylcholine location, levels and synthesis.

Aberrant Co-localization of Synaptic Proteins Promoted by Alzheimer's Disease Amyloid-ß Peptides: Protective Effect of Human Serum Albumin.

Amyloid-ß (Aß), Aß40, Aß42, and, recently, Aß25-35 have been directly implicated in the pathogenesis of Alzheimer's disease. We have studied the effects of Aß on neuronal death, reactive oxygen species (ROS) production, and synaptic assembling in neurons in primary culture. Aß25-35, Aß40, and Aß42 significantly decreased neuronal viability, although Aß25-35 showed a higher effect. Aß25-35 showed a more penetrating ability to reach mitochondria while Aß40 did not enter the neuronal cytosol and Aß42 was scarcely internalized. We did not observe a direct correlation between ROS production and cell death because both Aß40 and Aß42 decreased neuronal viability but Aß40 did not change ROS production. Rather, ROS production seems to correlate with the penetrating ability of each Aß. No significant differences were found between Aß40 and Aß42 regarding the extent of the deleterious effects of both peptides on neuronal viability or synaptophysin expression. However, Aß40 elicited a clear delocalization of PSD-95 and synaptotagmin from prospective synapsis to the neuronal soma, suggesting the occurrence of a crucial effect of Aß40 on synaptic disassembling. The formation of Aß40- or Aß42-serum albumin complexes avoided the effects of these peptides on neuronal viability, synaptophysin expression, and PSD-95/synaptotagmin disarrangement suggesting that sequestration of Aß by albumin prevents deleterious effects of these peptides. We can conclude that Aß borne by albumin can be safely transported through body fluids, a fact that may be compulsory for Aß disposal by peripheral tissues.

A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity.

The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43266-283, inhibits Src activity, we investigated the effect of TAT-Cx43266-283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43266-283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266-283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266-283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266-283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266-283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266-283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43266-283 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43266-283 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation.

Spoligotype diversity and 5-year trends of bovine tuberculosis in Extremadura, southern Spain.

Bovine tuberculosis (bTB) causes significant losses to farming economies worldwide. A better understanding on the epidemiology of this disease and the role that the different hosts develop in the maintenance and spread of bTB is vital to control this zoonotic disease. This study reports the spoligotype diversity and temporal evolution of Mycobacterium tuberculosis Complex (MTBC) isolates obtained from Extremadura (southern Spain). Genotyping data of Mycobacterium bovis (n = 2102) and Mycobacterium caprae (n = 96) isolates from cattle and wildlife species, collected between 2008 and 2012, were used in this study. The isolates resulted clustered into 88 spoligotypes which varied largely in frequency and occurrence in the three hosts. The 20 most frequent patterns represented 91.99 % of the isolates, the spoligotype SB0121 being the clearly predominant and most widely dispersed geographically. The major variety of the spoligotype patterns (78 out of 88) was isolated from the cattle, in fact 50 (56.83 %) of the patterns were found only in this species. Within the spoligotypes shared between the cattle and wildlife species, 17 patterns (1747 isolates) were shared with wild boar and Iberian red deer, 10 patterns (308 isolates) were exclusively shared with wild boar, and only one pattern (two isolates) was shared exclusively with Iberian red deer. The significant number of spoligotypes shared between the three hosts (79.49 %) highlights the components of the multi-host system that allows the bTB maintenance in our study area. The greater percentage of isolates shared by the wild boar and cattle (93.50 %) supports the role of wild boar as main maintenance host for bTB in cattle. These results could be extrapolated to areas with a similar epidemiological scenario and could be helpful for other countries where wild reservoirs represent a handicap for the successful eradication of bTB from livestock.