Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues

Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC:uidA, rolC:iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.     

Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells

The cytotoxic and mutagenic effect of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excision diploid human cells treated just prior to onset of S was compared with that of cells allowed ～ 16 h for excision repair before onset of S and with that observed in excision-deficient serodema pigmentosum (SP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began ～ 22 h after the cells were plated at lower density (i.e., 1.4 × 10⁴ cells/cm²). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was ～ 12- to 16-fold higher than that observed in cells treated in early G₁ or treated in G₀ (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G₁ or in G₀, No such difference could be seen in XP12BE cells treated in exponential growth or in G₀. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells give a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period priot to S support the conclusions.     

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

Alternative complex formation of the Ca-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis

Intracellular release of calcium ions belongs to the earliest events in cellular stress perception. The molecular mechanisms integrating signals from different environmental cues and translating them into an optimized response are largely unknown. We report here the functional characterization of CIPK1, a protein kinase interacting strongly with the calcium sensors CBL1 and CBL9. Comparison of the expression patterns indicates that the three proteins execute their functions in the same tissues. Physical interaction of CIPK1 with CBL1 and CBL9 targets the kinase to the plasma membrane. We show that, similarly to loss of CBL9 function, mutation of either CBL1 or CIPK1 renders plants hypersensitive to osmotic stress. Remarkably, in contrast to the cbl1 mutant and similarly to the cbl9 mutant, loss of CIPK1 function impairs abscisic acid (ABA) responsiveness. We therefore suggest that, by alternative complex formation with either CBL1 or CBL9, the kinase CIPK1 represents a convergence point for ABA-dependent and ABA-independent stress responses. Based on our genetic, physiological and protein-protein interaction data, we propose a general model for information processing in calcium-regulated signalling networks.     

Structural insights into dehydratase substrate selection for the borrelidin and fluvirucin polyketide synthases

Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,β-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B₁ PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3–β11 and β7–α2. From the catalytic Asp located in α3 to a conserved Pro in β11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.

     

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.     

The acute systemic toxicity of thallium in rats produces oxidative stress: attenuation by metallothionein and Prussian blue

BioMetals - Thallium (TI) is one of the most toxic heavy metals. Human exposure to Tl occurs through contaminated drinking water and from there to food, a threat to health. Recently, environmental...