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.

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.

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.

Expanding the Strained Alkyne Toolbox: Generation and Utility of Oxygen-Containing Strained Alkynes.

We report synthetic methodology that permits access to two oxacyclic strained intermediates, the 4,5-benzofuranyne and the 3,4-oxacyclohexyne. In situ trapping of these intermediates affords an array of heterocyclic scaffolds by the formation of one or more new C-C or C-heteroatom bonds. Experimentally determined regioselectivities were consistent with predictions made using the distortion/interaction model and were also found to be greater compared to selectivities seen in the case of trapping experiments of the corresponding N-containing intermediates. These studies demonstrate the synthetic versatility of oxacyclic arynes and alkynes for the synthesis of functionalized heterocycles, while further expanding the scope of the distortion/interaction model. Moreover, these efforts underscore the value of harnessing strained heterocyclic intermediates as a unique approach to building polycyclic heteroatom-containing frameworks.

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.

Generation and regioselective trapping of a 3,4-piperidyne for the synthesis of functionalized heterocycles.

We report the generation of the first 3,4-piperidyne and its use as a building block for the synthesis of annulated piperidines. Experimental and computational studies of this intermediate are disclosed, along with comparisons to the well-known 3,4-pyridyne. The distortion/interaction model is used to explain the observed regioselectivities.

Classification of peacock feather reflectance using principal component analysis similarity factors from multispectral imaging data.

Iridescent structural colors in biology exhibit sophisticated spatially-varying reflectance properties that depend on both the illumination and viewing angles. The classification of such spectral and spatial information in iridescent structurally colored surfaces is important to elucidate the functional role of irregularity and to improve understanding of color pattern formation at different length scales. In this study, we propose a non-invasive method for the spectral classification of spatial reflectance patterns at the micron scale based on the multispectral imaging technique and the principal component analysis similarity factor (PCASF). We demonstrate the effectiveness of this approach and its component methods by detailing its use in the study of the angle-dependent reflectance properties of Pavo cristatus (the common peacock) feathers, a species of peafowl very well known to exhibit bright and saturated iridescent colors. We show that multispectral reflectance imaging and PCASF approaches can be used as effective tools for spectral recognition of iridescent patterns in the visible spectrum and provide meaningful information for spectral classification of the irregularity of the microstructure in iridescent plumage.

The role of aryne distortions, steric effects, and charges in regioselectivities of aryne reactions.

The distortion/interaction model has been used to explain and predict reactivity in a variety of reactions where more common explanations, such as steric and electronic factors, do not suffice. This model has also provided new fundamental insight into regioselectivity trends in reactions of unsymmetrical arynes, which in turn has fueled advances in aryne methodology and natural product synthesis. This article describes a systematic experimental and computational study of one particularly important class of arynes, 3-halobenzynes. 3-Halobenzynes are useful synthetic building blocks whose regioselectivities have been explained by several different models over the past few decades. Our efforts show that aryne distortion, rather than steric factors or charge distribution, are responsible for the regioselectivities observed in 3-haloaryne trapping experiments. We also demonstrate the synthetic utility of 3-halobenzynes for the efficient synthesis of functionalized heterocycles, using a tandem aryne-trapping/cross-coupling sequence involving 3-chlorobenzyne.

Cycloadditions of cyclohexynes and cyclopentyne.

We report the strategic use of cyclohexyne and the more elusive intermediate, cyclopentyne, as a tool for the synthesis of new heterocyclic compounds. Experimental and computational studies of a 3-substituted cyclohexyne are also described. The observed regioselectivities are explained by the distortion/interaction model.

Isolation of Aeromonas hydrophila in the respiratory tract of wild boar: pathologic implications.

Aeromonas hydrophila has been repeatedly reported as an animal pathogen. This study describes a case of a wild boar piglet in Spain with severe purulent pneumonia caused by A. hydrophila. To confirm the presence of A. hydrophila in the respiratory tract of wild boars from the same region, lung samples from 34 adult hunted animals and nasal samples from 12 live animals were collected and cultured in selective medium. Lung lesions were studied in hunted wild boars to determine the presence of A. hydrophila and to assess its role as a possible respiratory pathogen in wild boars. A. hydrophila was isolated in 10.87% of the animals studied (8.82% of the dead animals and 16% of the live animals). However, its presence in the lungs of adults could not be correlated with the lesions found. Thus, the role of A. hydrophila as a respiratory pathogen is likely to be influenced by other factors, such as age, immunologic status, or coinfection with other pathogens. As a zoonotic pathogen, the presence of A. hydrophila in wild boars may pose a potential risk to people who consume their flesh.

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.

Reduced connexin43 expression correlates with c-Src activation, proliferation, and glucose uptake in reactive astrocytes after an excitotoxic insult.

In diverse brain pathologies, astrocytes become reactive and undergo profound phenotypic changes. Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is one of the proteins modified in reactive astrocytes. Downregulation of Cx43 in cultured astrocytes activates c-Src, promotes proliferation, and increases the rate of glucose uptake; however, so far there have been no studies examining whether this cascade of events takes place in reactive astrocytes. In this work, we analyzed this pathway after a cortical lesion induced by a kainic acid injection. As previously described, astrocytes reacted to the lesion with an increase in glial fibrillary acidic protein and a decrease in Cx43 expression. Some of these reactive astrocytes proliferated, as estimated by bromodeoxyuridine incorporation and cyclins D1 and D3 upregulation. In addition, the expression of the glucose transporter GLUT-3 and the enzyme responsible for glucose phosphorylation, Type II hexokinase (Hx-2), were induced in reactive astrocytes, suggesting an increased glucose uptake. Previous in vitro studies reported that c-Src is the link between Cx43 and glucose uptake and proliferation in astrocytes. Here, we found that c-Src activity increased in the lesioned area. c-Src activation and Cx43 downregulation preceded the peak of Hx-2 and cyclin D3 expression, suggesting that c-Src could mediate the effect of Cx43 on glucose uptake and proliferation in reactive astrocytes after an excitotoxic insult. Interestingly, we identify c-Src, GLUT-3, and Hx-2 in the signaling mechanisms involved in the reaction of astroglia to injury. Altogether these data contribute to identify new therapeutical targets to enhance astrocyte neuroprotective activities.

Low-frequency correlations (1/f(α) type) in paint application of metallic colors.

We examine trial-to-trial variability of color coordinates in automotive coatings containing effect pigments, which are considered a reference paradigm for engineering angle-dependent color effects. We report the existence of correlations that show 1/f - Fourier spectra at low frequencies in all color coordinates. The scaling exponent was lower at near-specular conditions for lightness variations, suggesting a contribution from the deposition of metal flakes in metallic colors. However, the exponent was lower near the specular for blue-yellow variations, suggesting a contribution from chemical pigments in solid colors. These results were independent of the illuminant spectra. The methods employed are useful in the evaluation of industrial color matching among assembly parts.

1/f Noise in human color vision: the role of S-cone signals.

We examine the functional role of S-cone signals on reaction time (RT) variability in human color vision. Stimuli were selected along red-green and blue-yellow cardinal directions and at random directions in the isoluminant plane of the color space. Trial-to-trial RT variability was not statistically independent but correlated across experimental conditions and exhibited 1/f noise spectra with an exponent close to unity in most of the cases. Regarding contrast coding, 1/f noise for random chromatic stimuli at isoluminance was similar to that for achromatic stimuli, thus suggesting that S-cone signals reduce variability of higher order color mechanisms. If we regard spatial coding, the effect of S-cone density in the retina on RT variability was investigated. The magnitude of 1/f noise at 16 min of arc (S-cone free zone) was higher than at 90 min of arc in the blue-yellow channel, and it was similar for the red-green channel. The results suggest that S-cone signals are beneficial and they modulate 1/f noise spectra at postreceptoral stages. The implications related to random multiplicative processes as a possible source of 1/f noise and the optimal information processing in color vision are discussed.

Scattering characterization of nanopigments in metallic coatings using hyperspectral optical imaging.

We have determined the reflectance spectra of colored metallic coatings with high spatial resolution by using a hyperspectral imaging system. Reflectance spectra were converted to color coordinates revealing characteristic color maps in the color space. Principal-component analysis was applied to decorrelate the spatial variability of the reflectance spectra. We found that the eigenvalue spectra follow different power laws. The scaling exponent was analyzed by considering random-walk-type processes. An estimation of the Hurst exponent was done, suggesting anomalous diffusion from multiple light scattering. The results show that hyperspectral imaging combined with principal-component analysis provides a valuable method for nondestructive testing of complex turbid media.

Oleic acid synthesized in the periventricular zone promotes axonogenesis in the striatum during brain development.

Our previous works showed that oleic acid synthesized in vitro by astrocytes in response to albumin behaves as a neurotrophic factor in neurons, up-regulating several proteins, such as the axonal growth marker growth-associated protein 43(GAP-43). Although the molecular mechanism of this process is fairly known, there is no evidence pinpointing the region/s in which oleic acid is synthesized. In this study, we show that the rate-limiting enzyme in oleic acid synthesis, stearoyl-CoA desaturase (SCD-1), is located in the periventricular zone of the brain of newborn rats, simultaneously to an increase in the amount of free oleic acid in the forebrain. In addition, the spatio-temporal presence of albumin - the signal that promotes oleic acid synthesis - and that of GAP-43 are correlated with that of SCD-1. Using organotypic slice cultures, we found that albumin up-regulates SCD-1 and stimulates the growth of GAP-43-positive axons in the striatum. The effect of albumin on GAP-43 was reduced when SCD-1 was silenced by siRNA. In conclusion, our results suggest that albumin up-regulates axonogenesis in the striatum by increasing the amount of the neurotrophic factor oleic acid synthesized by SCD-1 in the periventricular zone of the newborn brain.

S-cone excitation ratios for reaction times to blue-yellow suprathreshold changes at isoluminance.

We examined different contrast metrics to scale visual latencies for suprathreshold stimuli modulated along tritan confusion lines. S-cone increments ('blue') and decrements ('yellow') were isolated along two different tritan confusion lines, each one having a different luminance value. Reaction times (RT) were evaluated as a function of the Weber contrast and the S-cone excitation ratio between the test stimulus and the background. RTs were described using a model that generalizes Piéron's law and incorporates the notion of threshold units and power law scaling. Our results show that RTs for S-cone increments and decrements equate better when using the S-cone excitation ratio. However, a single function did not describe all RT data. S-cone RTs are better described by separate functions. We conclude that S-cone increments and decrements do not scale in the same manner. Both Weber contrast and the S-cone excitation ratio are plausible metrics at isoluminance. The implications for the S-cone pathways are discussed.

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.

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).