Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of ...the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.
Pacific sand lance Ammodytes personatus are a key forage fish in the North Pacific for many species of salmon, groundfish, seabirds, and marine mammals and have historically been important to ...predators in relatively warm years. However, extreme declines in the nutritional value of sand lance in Prince William Sound, Alaska, USA, during 2012–2016 indicate that energy transfer from lower trophic levels to predators via sand lance may have been disrupted during the North Pacific marine heatwave in 2015 and 2016. Nutritional value (length, energy density, and whole-body energy) was measured in age-0 and age-1 sand lance collected during July in cool (2012–2013) and increasingly warm (2014–2016) years. The value of age-0 fish was relatively stable, with only minor differences among years for length and whole-body energy. By contrast, the value of age-1 fish significantly declined in 2015, and by 2016 they were 38% shorter and 13% lower in energy density compared to cooler years. This contributed to significant declines in whole-body energy of 44% in 2015 and 89% in 2016 compared to cooler years (2012–2014). The 2015 sand lance cohort experienced little growth or lipid accumulation from July 2015 at age-0 to July 2016 at age-1. This effective disruption of energy flow through pelagic food webs probably contributed to population declines and/or breeding failures observed among several predators in the Gulf of Alaska and suggests that tipping points were reached during the heatwave.
A protracted period (2014-2016) of anomalously warm water in the northeast Pacific Ocean precipitated an extensive die-off of common murres Uria aalge (hereafter ‘murres’) during 2015-2016, ...accompanied by reduced colony attendance and reproductive success of murres and black-legged kittiwakes Rissa tridactyla (‘kittiwakes’) starting in 2015. Most murres died of starvation following a large-scale reduction in abundance and quality of forage fish. To assess murre and kittiwake recovery following the marine heatwave, we monitored their demographics at 2 colonies (Chisik and Gull Islands) in Cook Inlet, Alaska (USA), from 2016 to 2019. Compared to historic data (1995-1999), we observed declines and increased variability in colony attendance and productivity across species and colonies, and predation was widespread. At Chisik, where food limitations were common during historic studies, both species experienced substantial population declines and reproductive failures in all 4 years (2016-2019) following the heatwave. At Gull, a typically productive colony during historic studies, murres failed to fledge chicks for 3 years (2016-2018) following the heatwave. By 2019, murre productivity recovered to about half that observed during historic studies (0.28 vs. 0.54 chicks per pair), but populations had declined by half. Kittiwake population size at Gull declined a quarter from historic counts, and reproduction alternated between complete breeding failures (2016/2018) and high productivity (2017/2019). These multi-year demographic impacts indicate lingering effects of the heatwave on kittiwakes and murres through forage fish depletion and increased predator disturbance, and possibly other stressors. It remains unknown whether populations can rebound to historic levels. If so, recovery would likely take decades.
In situ heart valve tissue engineering is an emerging approach in which resorbable, off-the-shelf available scaffolds are used to induce endogenous heart valve restoration. Such scaffolds are ...designed to recruit endogenous cells in vivo, which subsequently resorb polymer and produce and remodel new valvular tissue in situ. Recently, preclinical studies using electrospun supramolecular elastomeric valvular grafts have shown that this approach enables in situ regeneration of pulmonary valves with long-term functionality in vivo. However, the evolution and mechanisms of inflammation, polymer absorption and tissue regeneration are largely unknown, and adverse valve remodeling and intra- and inter-valvular variability have been reported. Therefore, the goal of the present study was to gain a mechanistic understanding of the in vivo regenerative processes by combining routine histology and immunohistochemistry, using a comprehensive sheep-specific antibody panel, with Raman microspectroscopy for the spatiotemporal analysis of in situ tissue-engineered pulmonary valves with follow-up to 24 months from a previous preclinical study in sheep. The analyses revealed a strong spatial heterogeneity in the influx of inflammatory cells, graft resorption, and foreign body giant cells. Collagen maturation occurred predominantly between 6 and 12 months after implantation, which was accompanied by a progressive switch to a more quiescent phenotype of infiltrating cells with properties of valvular interstitial cells. Variability among specimens in the extent of tissue remodeling was observed for follow-up times after 6 months. Taken together, these findings advance the understanding of key events and mechanisms in material-driven in situ heart valve tissue engineering.
This study describes for the first time the long-term in vivo inflammatory and regenerative processes that underly in situ heart valve tissue engineering using resorbable synthetic scaffolds. Using a unique combinatorial analysis of immunohistochemistry and Raman microspectroscopy, important spatiotemporal variability in graft resorption and tissue formation was pinpointed in in situ tissue-engineered heart valves, with a follow-up time of up to 24 months in sheep. This variability was correlated to heterogenous regional cellular repopulation, most likely instigated by region-specific differences in surrounding tissue and hemodynamics. The findings of this research contribute to the mechanistic understanding of in situ tissue engineering using resorbable synthetics, which is necessary to enable rational design of improved grafts, and ensure safe and robust clinical translation.
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Previous tissue engineering approaches to create heart valves have been limited by the structural immaturity and mechanical properties of the valve constructs. This study used an in vitro pulse ...duplicator system to provide a biomimetic environment during tissue formation to yield more mature implantable heart valves derived from autologous tissue.
Trileaflet heart valves were fabricated from novel bioabsorbable polymers and sequentially seeded with autologous ovine myofibroblasts and endothelial cells. The constructs were grown for 14 days in a pulse duplicator in vitro system under gradually increasing flow and pressure conditions. By use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and the valve constructs were implanted into 6 lambs (weight 19+/-2.8 kg). All animals had uneventful postoperative courses, and the valves were explanted at 1 day and at 4, 6, 8, 16, and 20 weeks. Echocardiography demonstrated mobile functioning leaflets without stenosis, thrombus, or aneurysm up to 20 weeks. Histology (16 and 20 weeks) showed uniform layered cuspal tissue with endothelium. Environmental scanning electron microscopy revealed a confluent smooth valvular surface. Mechanical properties were comparable to those of native tissue at 20 weeks. Complete degradation of the polymers occurred by 8 weeks. Extracellular matrix content (collagen, glycosaminoglycans, and elastin) and DNA content increased to levels of native tissue and higher at 20 weeks.
This study demonstrates in vitro generation of implantable complete living heart valves based on a biomimetic flow culture system. These autologous tissue-engineered valves functioned up to 5 months and resembled normal heart valves in microstructure, mechanical properties, and extracellular matrix formation.
Purpose
Prior reports on the location and sizes of brain metastases almost entirely focus on patients with primary breast and pulmonary cancer. This is the first study comparing multiple other types ...of cancer that metastasize to the brain.
Methods
This monocentric retrospective study includes 369 untreated patients with 3313 intraaxial brain metastases. Following semi-manual segmentation of metastases on post-contrast T1WI, cumulative spatial probability distribution maps of brain metastases were created for the whole group and for all primary tumors. Furthermore, mixed effects logistic regression model analysis was performed to determine if the primary tumor, patient age, and patient sex influence lesion location.
Results
The cerebellum as location of brain metastases was proportionally overrepresented. Breast and pulmonary cancer caused higher number of brain metastases to what would normally be expected. Multivariate analyses revealed a significant accumulation of brain metastases from skin cancer in a frontal and from breast and gastrointestinal cancer in a cerebellar location.
Conclusion
Distribution of brain metastases is very heterogeneous for the distinct primaries, possibly reflecting the diversity of mechanisms involved in brain metastases formation. In daily clinical practice distribution patters may be beneficial to predict the primary cancer site, if unknown.
Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to ...physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The mechanisms of extracellular matrix changes accompanying myxomatous valvular degeneration are uncertain.
To test the hypothesis that valvular interstitial cells mediate extracellular matrix ...degradation in myxomatous degeneration by excessive secretion of catabolic enzymes, we examined the functional characteristics of valvular interstitial cells in 14 mitral valves removed for myxomatous degeneration from patients with mitral regurgitation and in 11 normal mitral valves obtained at autopsy. Immunohistochemical staining assessed (1) cell phenotype using antibodies to alpha-actin (microfilaments), vimentin and desmin (intermediate filaments), smooth muscle myosin (SM1), and SMemb (a nonmuscle myosin produced by activated mesenchymal cells) and (2) the expression of proteolytic activity using antibodies to collagenases (matrix metalloproteinase MMP-1, MMP-13), gelatinases (MMP-2, MMP-9), cysteine endoproteases (cathepsin S and K), and interleukin-1beta, a cytokine that can induce secretion of proteolytic enzymes. Although interstitial cells in normal valves stained positively for vimentin, but not alpha-actin or desmin, cells in myxomatous valves contained both vimentin and alpha-actin or desmin (characteristics of myofibroblasts). Moreover, cells in myxomatous valves strongly expressed SMemb, MMPs, cathepsins, and interleukin-1beta, which were weakly stained in controls. Nevertheless, interstitial cells in both groups strongly expressed procollagen-I mRNA (in situ hybridization), suggesting preserved ability to synthesize collagen in myxomatous valves.
Interstitial cells in myxomatous valves have features of activated myofibroblasts and express excessive levels of catabolic enzymes, without altered levels of interstitial collagen mRNA. We conclude that valvular interstitial cells regulate matrix degradation and remodeling in myxomatous mitral valve degeneration.
Unstable atherosclerotic plaques that cause acute coronary events usually contain abundant macrophages expressing matrix metalloproteinases (MMPs) and tissue factor (TF), molecules that probably ...contribute to plaque rupture and subsequent thrombus formation. Lipid lowering with HMG-CoA reductase inhibitors reduces acute coronary events.
To test whether lipid lowering with an HMG-CoA reductase inhibitor retards macrophage accumulation in rabbit atheroma, we administered cerivastatin to immature Watanabe heritable hyperlipidemic rabbits (cerivastatin group, n=10, cerivastatin 0.6 mg x kg(-1) x d(-1); control group, n=9, saline 0.6 mL x kg(-1) x d(-1)) for 32 weeks and measured macrophage accumulation and expression of MMPs and TF. Serum cholesterol levels after 32 weeks were 809+/-40 mg/dL (control group) and 481+/-24 mg/dL (treated group). Cerivastatin diminished accumulation of macrophages in aortic atheroma. Macrophage expression of MMP-1, MMP-3, MMP-9, and TF also decreased with cerivastatin treatment. Cerivastatin reduced the number of macrophages expressing histone mRNA (a sensitive marker of cell proliferation) detected by in situ hybridization but did not alter macrophages bearing a marker of death (TUNEL staining). Cerivastatin treatment (>or=0.01 micromol/L) also reduced growth, proteolytic activity due to MMP-9, and TF expression in cultured human monocyte/macrophages.
These results suggest that lipid lowering with HMG-CoA reductase inhibitors alters plaque biology by reducing proliferation and activation of macrophages, prominent sources of molecules responsible for plaque instability and thrombogenicity.
The expression patterns of the recently discovered family of semaphorin genes suggests that they have widespread roles in embryonic development. Some seem to guide neuronal growth cones, but ...otherwise their functions are unknown. Semaphorin III is a membrane-associated secreted protein with a developmentally dynamic pattern of expression, including particular domains of the nervous system, the borders of developing bones, and the heart. In vitro, semaphorin III causes growth-cone collapse, and repels cutaneous sensory axons from the ventral spinal cord. Mutants in the Drosophila gene semaII, which encodes a related semaphorin, die after eclosion, but no responsible abnormality is evident. We have generated mice mutant in the semaIII gene by homologous recombination. Here we show that in the mutants, some sensory axons project into inappropriate regions of the spinal cord where semaIII is normally expressed. The cerebral cortex of homozygous mutant mice shows a paucity of neuropil and abnormally oriented neuronal processes, especially of the large pyramidal neurons. Certain embryonic bones and cartilaginous structures develop abnormally, with vertebral fusions and partial rib duplications. The few mice that survive more than a few days postnatally manifest pronounced and selective hypertrophy of the right ventricle of the heart and dilation of the right atrium. Thus, semaphorin III might serve as a signal that restrains growth in several developing organs.
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