Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Factors Released From Embryonic Stem Cells Stimulate C-Kit-Flk-1 \u3csup\u3e+Ve\u3c/sup\u3e Progenitor Cells And Enhance Neovascularization

We examined whether factors released from embryonic stem (ES) cells inhibit cardiac and vascular cell apoptosis and stimulate endogenous progenitor cells that enhance neovascularization with improved cardiac function. We generated and transplanted ES-conditioned medium (CM) in the infarcted heart to examine effects on cardiac and vascular apoptosis, activation of endogenous c-kit and FLK-1+ve cells, and their role in cardiac neovascularization. TUNEL, caspase-3 activity, immunohistochemistry, H&E, and Masson\u27s trichrome stains were used to determine the effect of transplanted ES-CM on cardiac apoptosis and neovascularization. TUNEL staining and caspase-3 activity confirm significantly (p\u3c0.05) reduced apoptosis in MI+ES-CM compared with MI+ cell culture medium. Immunohistochemistry demonstrated increased (p\u3c0.05, 53%) c-kit+ve and FLK-1+ve positive cells, as well as increased (p\u3c0.05, 67%) differentiated CD31-positive cells in ES-CM groups compared with respective controls. Furthermore, significantly (p\u3c0.05) increased coronary artery vessels were observed in ES-CM transplanted hearts compared with control. Heart function was significantly improved following ES-CM transplantation. Next, we observed significantly increased (p\u3c0.05) levels of c-kit activation proteins (HGF and IGF-1), anti-apoptosis factors (IGF-1 and total antioxidants), and neovascularization protein (VEGF). In conclusion, we suggest that ES-CM following transplantation in the infarcted heart inhibits apoptosis, activates cardiac endogenous c-kit and FLK-1+ve cells, and differentiates them into endothelial cells (ECs) that enhances neovascularization with improved cardiac function. © 2010 Mary Ann Liebert, Inc

Factors Released from Embryonic Stem Cells Stimulate c-kit-FLK-1+ve Progenitor Cells and Enhance Neovascularization

We examined whether factors released from embryonic stem (ES) cells inhibit cardiac and vascular cell apoptosis and stimulate endogenous progenitor cells that enhance neovascularization with improved cardiac function. We generated and transplanted ES-conditioned medium (CM) in the infarcted heart to examine effects on cardiac and vascular apoptosis, activation of endogenous c-kit and FLK-1+ve cells, and their role in cardiac neovascularization. TUNEL, caspase-3 activity, immunohistochemistry, H&E, and Masson's trichrome stains were used to determine the effect of transplanted ES-CM on cardiac apoptosis and neovascularization. TUNEL staining and caspase-3 activity confirm significantly (p < 0.05) reduced apoptosis in MI+ES-CM compared with MI+ cell culture medium. Immunohistochemistry demonstrated increased (p < 0.05, 53%) c-kit+ve and FLK-1+ve positive cells, as well as increased (p < 0.05, 67%) differentiated CD31-positive cells in ES-CM groups compared with respective controls. Furthermore, significantly (p < 0.05) increased coronary artery vessels were observed in ES-CM transplanted hearts compared with control. Heart function was significantly improved following ES-CM transplantation. Next, we observed significantly increased (p < 0.05) levels of c-kit activation proteins (HGF and IGF-1), anti-apoptosis factors (IGF-1 and total antioxidants), and neovascularization protein (VEGF). In conclusion, we suggest that ES-CM following transplantation in the infarcted heart inhibits apoptosis, activates cardiac endogenous c-kit and FLK-1+ve cells, and differentiates them into endothelial cells (ECs) that enhances neovascularization with improved cardiac function. Antioxid. Redox Signal. 13, 1857–1865

Reconstructing neuronal anatomy from whole-brain images

Reconstructing multiple molecularly defined neurons from individual brains and across multiple brain regions can reveal organizational principles of the nervous system. However, high resolution imaging of the whole brain is a technically challenging and slow process. Recently, oblique light sheet microscopy has emerged as a rapid imaging method that can provide whole brain fluorescence microscopy at a voxel size of 0.4\times 0.4\times 2.5\mu \mathrm{m}^{3}. On the other hand, complex image artifacts due to whole-brain coverage produce apparent discontinuities in neuronal arbors. Here, we present connectivity-preserving methods and data augmentation strategies for supervised learning of neuroanatomy from light microscopy using neural networks. We quantify the merit of our approach by implementing an end-to-end automated tracing pipeline. Lastly, we demonstrate a scalable, distributed implementation that can reconstruct the large datasets that sub-micron whole-brain images produce. © 2019 IEEE

The Drosophila Duox maturation factor is a key component of a positive feedback loop that sustains regeneration signaling.

Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth

Fatty Acid Profile and Biological Data of Four Endemic Cephalaria Species Grown in Turkey

WOS: 000299683800008The fatty acid compositions of the n-hexane extracts of the aerial parts of four Turkish Cephalaria species (C. paphlagonica, C. stellipilis, C. davisiana and C. elazigensis var. purpurea) were analyzed by GC-MS for the first time. The oil yields of these species were determined as ranging from 0.07% to 0.36 %. Seventeen fatty acids as methyl esters were identified. All extracts were found to contain significant quantities of palmitic, linoleic (LA), stearic and alpha-linolenic acid (ALA). ALA was the most abundant fatty acid in all species (29.00%, 30.51%, 32.49% and 34.87% for C. stellipilis, C. elazigensis, C. davisiana and C. paphlagonica, respectively). Other dominant fatty acid was palmitic acid, which ranged from 19.10% to 28.23% for all species. LA was detected in a considerable amount of 19.44 % for C. paphlagonica. The n-hexane extracts of the plants were also checked for their antimicrobial and antioxidant activities.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [107T028]The authors acknowledge to TUBITAK for financial support (107T028) and also Hygiene Center of Izmir for GC-MS analyses

Bioactive compounds, antioxidant and antimicrobial activities of Arum maculatum leaves extracts as affected by various solvents and extraction methods

The different species of Arum maculatum plant can be found in all over the world, and a wide range of medicinal applications has been mentioned for them. Thus, it can also be valued as a source of natural compounds with antioxidant and antimicrobial activities. In this study, the effect of solvents (water, ethanol, ethanol:water (50:50)) and extraction methods (maceration and ultrasound) on the extraction yields and bioactive properties of extracts were analyzed. The antioxidant capacity of Arum maculatum leaves extracts was investigated, and the concentrations of total phenolics, tocopherols, tannins and flavonoids were determined. 1,1‐diphenyl 2‐picrylhydrazyl free radical (DPPH), β‐Carotene bleaching, and oxidative stability index (OSI) were used to determine antioxidant activity. The ability to scavenge radicals was measured in these experiments by the discoloration of the solution. Also, the antimicrobial activity of different extracts against Gram‐positive (Staphylococcus aureus and Listeria monocytogenes) and Gram‐negative bacteria (Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa) was evaluated by using of microdilution and agar diffusion assays. The results demonstrated that ultrasonic extracts (especially ethanol:water (50:50) solvent) had the higher extraction yield and antioxidant potential than maceration extracts. All extracts were effective against all tested bacteria, and Listeria monocytogenes was the most sensitive bacterium with lowest MIC value (12.5 mg/ml) and biggest diameter of growth inhibition zone (13.77 mm). Generally, this Arum maculatum leaves extracts can be suggested as an economical source of antioxidant and antimicrobial agents and can be a suitable substitute for artificial and chemical food preservatives

NIP is required to sustain JNK signaling during late regeneration.

<p><b>(</b>A-H) Confocal images of fluorescence from the <i>TRE-red</i> reporter for JNK signaling in <i>w</i>^<i>1118</i> (A-D) and <i>mol</i>^{<i>e02670</i>}<i>/+</i> (E-H) regenerating discs at R0 (A,B), R24 (B,F), R48 (C,G) and R72 (D,H). (I) Quantification of fluorescence intensity of the <i>TRE-red</i> reporter in max projections of the confocal images at R0, because at this time point the epithelium cannot be distinguished from the debris. <i>w</i>^<i>1118</i> n = 10 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 14 discs. (J) Quantification of fluorescence intensity of the <i>TRE-red</i> reporter in single slices of the confocal images through the regenerating epithelium at R24, R48, and R72. R24 <i>w</i>^<i>1118</i> n = 11 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 11 discs. R48 <i>w</i>^<i>1118</i> = 14 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 15 discs. R72 <i>w</i>^<i>1118</i> n = 11 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 11 discs. (K) Regeneration assays using adult wing size to assess extent of regenerative growth in the imaginal discs in <i>w</i>^<i>1118</i>, <i>mol</i>^{<i>e02670</i>}<i>/+</i>, <i>puc</i>^<i>E69</i><i>/+</i>, and <i>mol</i>^{<i>e02670</i>}<i>/+;puc</i>^<i>E69</i><i>/+</i> animals. Two independent experiments, thus error bars are SD. <i>w</i>^<i>1118</i> n = 26 wings, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 83 wings, <i>puc</i>^<i>E69</i><i>/+</i> n = 99 wings, and <i>mol</i>^{<i>e02670</i>}<i>/+;puc</i>^<i>E69</i><i>/+</i> n = 95 wings. p<0.0001 for all comparisons using a chi-squared test. Dashed blue line outlines the wing primordium. Scale bars are 100 μm. Error bars are SEM unless otherwise noted. *p<0.05, **p<0.001, ***p<0.0001.</p