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Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of DNA direct repeats found in many prokaryotic genomes. It was discovered in bacteria as their (adaptive) immune system against invading viruses. Cas9...
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Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of DNA direct repeats found in many prokaryotic genomes. It was discovered in bacteria as their (adaptive) immune system against invading viruses. Cas9 is an endonuclease enzyme linked with the CRISPR system in bacteria. Bacteria use the Cas9 enzyme to chop viral DNA sequences by unwinding it and then finding the complementary base pairs to the guide RNA. CRISPR/Cas9 is a modern and powerful molecular biology approach that is widely used in genome engineering (to activate/repress gene expression). It can be used in vivo to cause targeted genome modifications with better efficiency as compared to meganucleases, zinc-finger nucleases and transcription activator-like effector nucleases. CRISPR/Cas9 is a simple, reliable, and rapid method for causing gene alterations that open new horizons of gene editing in a variety of living organisms, including humans, for the treatment of several diseases. In this short review, we explored the basic mechanisms underlying its working principles along with some of its current applications in a number of diverse fields.
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The trace element composition of planktic foraminiferal calcite provides a useful means of determining past surface ocean conditions. We have assembled the results of culture experiments for three species of symbiont-bearing plank...
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The trace element composition of planktic foraminiferal calcite provides a useful means of determining past surface ocean conditions. We have assembled the results of culture experiments for three species of symbiont-bearing planktic foraminifera, Globigerinoides ruber, Globigerinoides sacculifer, and Orbulina universa, and one symbiont-barren species, Globigerina bulloides, to evaluate their responses to temperature, salinity, pH, carbonate ion, and dissolved inorganic carbon (DIC) growth conditions. Trace element ratios (Li/Ca, B/Ca, Mg/Ca, Sr/Ca, Mn/Ca, Cd/Ca, Ba/Ca, Na/Ca, and U/Ca) were measured simultaneously on samples grown with the same culture techniques, which provides robust, relatable calibrations that may be used together in multi-proxy paleoceanographic studies. Our data confirm that temperature is the dominant control on foraminiferal Mg/Ca under the ranges of conditions studied and that the potential effects of salinity and CO32- on Mg/Ca of these tropical species across late Pleistocene glacial cycles are relatively small. Carbonate system experiments suggest that Sr/Ca may be useful for reconstructing large DIC changes. Na/Ca increases with salinity in G. ruber (pink), but not in G. sacculifer. As these emerging proxy relationships become more firmly established, the synthesis of multiple trace element ratios may help paleoceanographers isolate the effects of different environmental parameters in paleo records. Calcification rates (mu g/day) vary among species and do not respond consistently to any experimental parameter. Comparison of our calcification rates with those observed in inorganic calcite precipitation experiments suggest that foraminifera calcify similar to 100x more slowly than inorganic calcites grown in similar solutions. We suggest that calcification rate does not typically exert a dominant control on trace element partitioning in planktic foraminiferal calcite, though it may play a role for some elements under certain circumstances. Differences in average growth rate cannot explain composition differences among species, pointing to alternative controls that may be biological in origin. (C) 2016 Elsevier Ltd. All rights reserved.
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In 1992, Ca2+ microdomains were shown to exist in presynaptic nerve terminals. Soon thereafter, in 1993, Ca2+ microdomains were demonstrated in the apical granule containing region of pancreatic acinar cells. The pancreatic acinar...
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In 1992, Ca2+ microdomains were shown to exist in presynaptic nerve terminals. Soon thereafter, in 1993, Ca2+ microdomains were demonstrated in the apical granule containing region of pancreatic acinar cells. The pancreatic acinar cell is specialized for bulk secretion of digestive enzymes and therefore has a relatively large apical micro-domain, dominated by secretory (zymogen) granules, in which Ca2+ signalling is of crucial physiological significance because of the need to exercise precise control of the exocytotic secretory events. Local Ca2+ signalling in the apical domain occurs by repetitive episodes of Ca2+ release from a relatively small volume of endoplasmic reticulum (ER) terminals that are functionally fully connected to the bulk of the ER in the baso-lateral region, which is the quantitatively dominant Ca2+ store. Thus Ca2+ release from the small volume of the apical ER terminals can be sustained by intra-ER Ca2+ diffusion from the basal to the apical parts of the cells. In this short review the particular characteristics of the apical Ca2+ signalling domain will be discussed with special emphasis on its passive and active Ca2+ buffering properties and its ability to respond to local Ca2+ elevations by Ca2+-induced Ca2+ release. The functional significance of these characteristics for appropriate Ca2+ spiking are discussed as well as the pathophysiological consequences of destroying the Ca2+ signalling microdomain. (C) 2015 Elsevier Ltd. All rights reserved.
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Introduction: The hippocampus is integral for learning and memory and is targeted by multiple diseases. Neuroimaging approaches frequently use hippocampal subfield volumes as a standard measure of neurodegeneration, thus making th...
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Introduction: The hippocampus is integral for learning and memory and is targeted by multiple diseases. Neuroimaging approaches frequently use hippocampal subfield volumes as a standard measure of neurodegeneration, thus making them an essential biomarker to study. Collectively, histologic parcellation studies contain various disagreements, discrepancies, and omissions. The present study aimed to advance the hippocampal subfield segmentation field by establishing the first histology based parcellation protocol, applied to n = 22 human hippocampal samples.Methods: The protocol focuses on five cellular traits observed in the pyramidal layer of the human hippocampus. We coin this approach the pentad protocol. The traits were: chromophilia, neuron size, packing density, clustering, and collinearity. Subfields included were CA1, CA2, CA3, CA4, prosubiculum, subiculum, presubiculum, parasubiculum, as well as the medial (uncal) subfields Subu, CA1u, CA2u, CA3u, and CA4u. We also establish nine distinct anterior-posterior levels of the hippocampus in the coronal plane to document rostrocaudal differences.Results: Applying the pentad protocol, we parcellated 13 subfields at nine levels in 22 samples. We found that CA1 had the smallest neurons, CA2 showed high neuronal clustering, and CA3 displayed the most collinear neurons of the CA fields. The border between presubiculum and subiculum was staircase shaped, and parasubiculum had larger neurons than presubiculum. We also demonstrate cytoarchitectural evidence that CA4 and prosubiculum exist as individual subfields.Discussion: This protocol is comprehensive, regimented and supplies a high number of samples, hippocampal subfields, and anterior-posterior coronal levels. The pentad protocol utilizes the gold standard approach for the human hippocampus subfield parcellation.
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The construction of a low affinity Ca2+-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca2+). The novel probe is a fusion of CEPIA3 with ezrin, and is used i...
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The construction of a low affinity Ca2+-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca2+). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca2+-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca2+ influx on intra-wrinkle Ca2+ in the macrophage cell line, RAW 264.7. During experimentally-induced Ca(2+)influx, EPIC3 reported Ca2+ concentrations at the cell cortex in the region of 30-50 mu M, with peak locations towards the tips of wrinkles reaching 80 mu M. These concentrations were asso-ciated with cleavage of ezrin (a substrate for the Ca2+ activated protease calpain-1) and released the C-terminal fluors. The cortical Ca2+ levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50-80 mu M) during phagocytosis. As phagocytosis was completed, hotspots of Ca2+ near the phagosome were also observed.
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Abstract An outbreak of the novel beta coronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) first came to light in December 2019, which has unfolded rapidly and turned out to be a global pandemic. Early progn...
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Abstract An outbreak of the novel beta coronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) first came to light in December 2019, which has unfolded rapidly and turned out to be a global pandemic. Early prognosis of viral contamination involves speedy intervention, disorder control, and good‐sized management of the spread of disease. Reverse transcription‐polymerase chain reaction, considered the gold standard test for detecting nucleic acids and pathogen diagnosis, provides high sensitivity and specificity. However, reliance on high‐priced equipped kits, associated reagents, and skilled personnel slow down sickness detection. Lately, the improvement of clustered regularly interspaced short palindromic repeat?(CRISPR)‐Cas (CRISPR‐associated protein)‐based diagnostic systems has reshaped molecular diagnosis due to their low cost, simplicity, speed, efficiency, high sensitivity, specificity, and versatility, which is vital for accomplishing point‐of‐care diagnostics. We reviewed and summarized CRISPR–Cas‐based point‐of‐care diagnostic strategies and research in these paintings while highlighting their characteristics and challenges for identifying SARS‐CoV‐2.
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Abundant CRISPR-Cas systems in nature provide us with unlimited valuable resources to develop a variety of versatile tools, which are powerful weapons in biological discovery and disease treatment. Here, we systematically review t...
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Abundant CRISPR-Cas systems in nature provide us with unlimited valuable resources to develop a variety of versatile tools, which are powerful weapons in biological discovery and disease treatment. Here, we systematically review the development of CRISPR-Cas based tools from DNA nuclease to RNA nuclease, from nuclease dependent-tools to nucleic acid recognition dependent-tools. Also, considering the limitations and challenges of current CRISPR-Cas based tools, we discuss the potential directions for development of novel CRISPR toolkits in the future.
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Abstract Background Serum tumor markers including AFU, AFP, CEA, CA199, CA125 and CA724, are of great importance in the diagnosis, prognostic prediction and recurrence monitoring of gastrointestinal malignancies. However, their si...
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Abstract Background Serum tumor markers including AFU, AFP, CEA, CA199, CA125 and CA724, are of great importance in the diagnosis, prognostic prediction and recurrence monitoring of gastrointestinal malignancies. However, their significance in gastric cancer (GC) patients with neoadjuvant therapy (NCT) is still uncertain. The aim of this study was to evaluate the predictive value of these six tumor markers in locally advanced GC patients who underwent NCT and curative surgery. Methods In total, 290 locally advanced GC patients who underwent NCT and D2 radical gastrectomy were retrospectively analyzed. Data on their tumor markers before (pre-) and after (post-) NCT and pathological characteristics were extracted from the database of our hospital. The optimal cutoff values of the six tumor markers were calculated by the ROC curve and Youden index. Their predictive significance was analyzed and survival curves for overall survival (OS) were obtained by the Kaplan-Meier method. Associations between categorical variables were explored by the chi-square test or Fisher’s exact test. Multivariate analyses were performed by the Cox regression model. Results Pre- and post-CA199, ?CA125 and -CA724 could predict overall survival (all P ??0.05). Conclusions CA724 is an independent factor for prognosis and could be used to predict ypN and ypTNM stage in locally advanced GC patients undergoing NCT and curative resection.
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Mitochondria have a very large capacity to accumulate Ca2+ during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca2+]M (mitochondrial [Ca2+]) may well reach millimolar levels in a few se...
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Mitochondria have a very large capacity to accumulate Ca2+ during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca2+]M (mitochondrial [Ca2+]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca2+]M during prolonged stimulation has been previously precluded by the high Ca2+ affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca2+] in the millimolar range for long periods of time without problems derived from aequorin consumption. We show in the present study that addition of Ca2+ to permeabilized HeLa cells triggers an increase in [Ca2+]M up to an steady state of approximately 2-3[NON-BREAKING SPACE]mM in the absence of phosphate and 0.5-1[NON-BREAKING SPACE]mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca2+] reaches 0.5-1[NON-BREAKING SPACE]mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca2+]M levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca2+ uniporter largely reduced the rate of [Ca2+]M increase, but the final steady-state [Ca2+]M reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca2+]M without problems derived from aequorin consumption.
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