Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and ...show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn's disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for the characterisation of macrophage polarisation in situ. Furthermore, CD163 cannot be considered a reliable M2 marker when used on its own.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We characterized wildfire transmission and exposure within a matrix of large land tenures (federal, state, and private) surrounding 56 communities within a 3.3 million ha fire prone region of central ...Oregon US. Wildfire simulation and network analysis were used to quantify the exchange of fire among land tenures and communities and analyze the relative contributions of human versus natural ignitions to wildfire exposure. Among the land tenures examined, the area burned by incoming fires averaged 57% of the total burned area. Community exposure from incoming fires ignited on surrounding land tenures accounted for 67% of the total area burned. The number of land tenures contributing wildfire to individual communities and surrounding wildland urban interface (WUI) varied from 3 to 20. Community firesheds, i.e. the area where ignitions can spawn fires that can burn into the WUI, covered 40% of the landscape, and were 5.5 times larger than the combined area of the community core and WUI. For the major land tenures within the study area, the amount of incoming versus outgoing fire was relatively constant, with some exceptions. The study provides a multi-scale characterization of wildfire networks within a large, mixed tenure and fire prone landscape, and illustrates the connectivity of risk between communities and the surrounding wildlands. We use the findings to discuss how scale mismatches in local wildfire governance result from disconnected planning systems and disparate fire management objectives among the large landowners (federal, state, private) and local communities. Local and regional risk planning processes can adopt our concepts and methods to better define and map the scale of wildfire risk from large fire events and incorporate wildfire network and connectivity concepts into risk assessments.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Entomopathogenic nematodes (EPNs) are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that ...EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs) of Steinernema carpocapsae (a well-studied EPN species) release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA ...sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
For half a century aqueous two‐phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they ...have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single‐use, flexible biomanufacturing has led to industrial re‐evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi‐stage operations and in single‐step purification processes.
Aqueous two‐phase systems (ATPS) are approached through a novel SWOT (Strengths, Weaknesses, Opportunities and Threats) perspective, where their potential for integration in industrial bioprocesses is evaluated based on classic knowledge and recent advances in this field. Overall, it can be concluded that ATPS weaknesses towards industrial implementation have been largely overcome, paving the way for opportunities in feed clarification, multi‐stage operation and single step purifications.
Large fires and their impacts are a growing concern as changes in climate and land use proceed. The study of large-fire controls remains incipient in comparison with other components of the fire ...regime. Improved understanding of large-fire size drivers can disclose fire–landscape relationships and inform more sustainable and effective fire management. We used boosted regression tree modeling to identify the variables influent on largefire size (100–23,219 ha, n = 609) in Portugal (1998–2008) and quantify their relative importance, globally and across the fire-size range. Potential explanatory variables included metrics pertaining to fire weather and antecedent rainfall, burned area composition, fuel connectivity, pyrodiversity (from fire recurrence patterns), topography, and land development. Large fires seldom occurred in the absence of severe fire weather. The fire-size model accounted for 70% of the deviance and included 12 independent variables, of which six absorbed 91% of the explanation. Bottom-up influences on fire size, essentially fuel-related, largely outweighed climate–weather influences, with respective importance of 85 and 15%. Fire size was essentially indifferent to land-cover composition, including forest type, and increased with high fuel connectivity and low pyrodiversity. Relevant synergies between variables were found, either positive or negative, for example, high pyrodiversity buffered the effects of extreme weather on fire size. The relative role of fire-size drivers did not vary substantially with fire size, but fires larger than 500 ha were increasingly controlled by fuel-related variables. The extent of an individual large fire is mainly a function of factors that land-use planning and forest and fuel management can tackle.
We study the dynamics of turbulent boundary layer flow over a heterogeneous topography composed of roughness patches exhibiting relatively high and low correlation in the streamwise and spanwise ...directions, respectively (i.e. the roughness appears as streamwise-aligned ‘strips’). It has been reported that such roughness induces a spanwise-wall normal mean secondary flow in the form of mean streamwise vorticity associated with counter-rotating boundary-layer-scale circulations. Here, we demonstrate that this mean secondary flow is Prandtl’s secondary flow of the second kind, both driven and sustained by spatial gradients in the Reynolds-stress components, which cause a subsequent imbalance between production and dissipation of turbulent kinetic energy that necessitates secondary advective velocities. In reaching this conclusion, we study (i) secondary circulations due to spatial gradients of turbulent kinetic energy, and (ii) the production budgets of mean streamwise vorticity by gradients of the Reynolds stresses. We attribute the secondary flow phenomena to extreme peaks of surface stress on the relatively high-roughness regions and associated elevated turbulence production in the fluid immediately above. An optimized state is attained by entrainment of fluid exhibiting the lowest turbulent stresses – from above – and subsequent lateral ejection in order to preserve conservation of mass.
Over the last years, aqueous two-phase systems (ATPS) regained an increasing interest due to their potential in the downstream processing of biomolecules. After many years with only a few articles ...published, a lot of effort and work has been put into studying these systems for the partitioning of a range of compounds including proteins, organic low-molecular weight molecules or metal ions. Although several research and review articles appeared, a background review on ATPS partitioning fundamentals is needed. In this article, partitioning theories and main effects of several important factors for partitioning, such as molecular weight of the polymer, effect of added salts, pH, electrical charges, and temperature on phase diagrams, tie-line lengths, interfacial tension and settling time of the two aqueous phases are extensively reviewed. The trend in ATPS research is given compiling the recent 2008-2013 research articles published in the field.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Background and Purpose
Immunomodulatory tetracyclines are well‐characterized drugs with a pharmacological potential beyond their antibiotic properties. Specifically, minocycline and doxycycline have ...shown beneficial effects in experimental colitis, although pro‐inflammatory actions have also been described in macrophages. Therefore, we aimed to characterize the mechanism behind their effect in acute intestinal inflammation.
Experimental Approach
A comparative pharmacological study was initially used to elucidate the most relevant actions of immunomodulatory tetracyclines: doxycycline, minocycline and tigecycline; other antibiotic or immunomodulatory drugs were assessed in bone marrow‐derived macrophages and in dextran sodium sulfate (DSS)‐induced mouse colitis, where different barrier markers, inflammatory mediators, microRNAs, TLRs, and the gut microbiota composition were evaluated. The sequential immune events that mediate the intestinal anti‐inflammatory effect of minocycline in DSS‐colitis were then characterized.
Key Results
Novel immunomodulatory activity of tetracyclines was identifed; they potentiated the innate immune response and enhanced resolution of inflammation. This is also the first report describing the intestinal anti‐inflammatory effect of tigecycline. A minor therapeutic benefit seems to derive from their antibiotic properties. Conversely, immunomodulatory tetracyclines potentiated macrophage cytokine release in vitro, and while improving mucosal recovery in colitic mice, they up‐regulated Ccl2, miR‐142, miR‐375 and Tlr4. In particular, minocycline initially enhanced IL‐1β, IL‐6, IL‐22, GM‐CSF and IL‐4 colonic production and monocyte recruitment to the intestine, subsequently increasing Ly6C−MHCII+ macrophages, Tregs and type 2 intestinal immune responses.
Conclusions and Implications
Immunomodulatory tetracyclines potentiate protective immune pathways leading to mucosal healing and resolution, representing a promising drug reposition strategy for the treatment of intestinal inflammation.
Magnetic separations in biotechnology Borlido, L.; Azevedo, A.M.; Roque, A.C.A. ...
Biotechnology advances,
12/2013, Letnik:
31, Številka:
8
Journal Article
Recenzirano
Magnetic separations are probably one of the most versatile separation processes in biotechnology as they are able to purify cells, viruses, proteins and nucleic acids directly from crude samples. ...The fast and gentle process in combination with its easy scale-up and automation provide unique advantages over other separation techniques. In the midst of this process are the magnetic adsorbents tailored for the envisioned target and whose complex synthesis spans over multiple fields of science. In this context, this article reviews both the synthesis and tailoring of magnetic adsorbents for bioseparations as well as their ultimate application.
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