scholarly journals A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

2020 ◽  
Author(s):  
Rachael E. Workman ◽  
Teja Pammi ◽  
Binh T. K. Nguyen ◽  
Leonardo W. Graeff ◽  
Erika Smith ◽  
...  
Keyword(s):  
Fold Increase ◽  
Bioinformatic Analysis ◽  
Type Ii ◽  
New Paradigm ◽  
Guide Rna ◽  
Immunization Rates ◽  
Regulatory Strategies ◽  
New Hosts ◽  
Intrinsic Regulation ◽  
The Cost

SUMMARYCRISPR-Cas systems provide their prokaryotic hosts with acquired immunity against viruses and other foreign genetic elements, but how these systems are regulated to prevent auto-immunity is poorly understood. In type II CRISPR-Cas systems, a transactivating CRISPR RNA (tracrRNA) scaffold functions together with a CRISPR RNA (crRNA) guide to program Cas9 for the recognition and cleavage of foreign DNA targets. Here, we show that a long-form tracrRNA performs an unexpected second function by folding into a natural single guide that directs Cas9 to transcriptionally repress its own promoter. Further, we demonstrate that Pcas9 serves as a critical regulatory node; de-repression causes a dramatic induction of Cas genes, crRNAs and tracrRNAs resulting in a 3,000-fold increase in immunization rates against unrecognized viruses. Heightened immunity comes at the cost of increased auto-immune toxicity, demonstrating the critical importance of the controller. Using a bioinformatic analysis, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

Pharmacoeconomical approaches for assessing the rational use of ophthalmic drugs in polyclinic settings

2021 ◽  
pp. 143-145
Author(s):  
N.A. Nefedov ◽  
◽  
D.S. Ramonas ◽  
B.G. Khasanov ◽  
A.S. Alexandrov ◽  
...  
Keyword(s):  
Fold Increase ◽  
Eye Drops ◽  
Ophthalmic Drugs ◽  
Rational Use ◽  
Rational Drug ◽  
Acquisition Costs ◽  
The Cost ◽  
Drug Provision ◽  
Combined Drugs

Purpose. To evaluate the results of the rational use of ophthalmic medicines with the use of pharmacoeconomical analysis. Material and methods. With the help of ABC-VEN analysis, a comparative study of the quality of pharmacotherapy and drug provision of patients who were monitored and treated by an ophthalmologist of the polyclinic in 2015 and 2019 was conducted. Results. As a result, it was found that the share of costs for vital drugs increased by 10.1% and amounted to 71.1% in 2019, which corresponds to the standardized criterion (70-80%). There was a reduction in the cost of purchasing secondary drugs by 7.6%, which indicates a rational drug supply of ophthalmic drugs. The most commonly used drugs for the treatment of glaucoma were 27.8% in 2015 and 35.1% in 2019. The share of their acquisition costs was 54.9% and 67.9%, respectively. There was a significant increase in the range and number of drugs for the treatment of patients with glaucoma: a 2.8-fold increase in the number of purchased eye drops for monotherapy and a 12% increase in the number of combined drugs. Conclusion. Pharmacoeconomical analysis showed an optimization of spending money on the purchase of drugs used in ophthalmology: an increase of 10.1% in the share of vital drugs and a decrease of 7.6% in the cost of purchasing secondary drugs. There was an increase in the range and quantity of drugs for the treatment of patients with glaucoma. Key words: ABC-VEN-analysis, drugs, ophthalmology, pharmacoeconomics.

Policy on the Development of Immunization Tracking Systems

PEDIATRICS ◽  
1996 ◽  
Vol 97 (6) ◽  
pp. 927-927
Author(s):  
Keyword(s):  
Health Care ◽  
Health Care Providers ◽  
Care Providers ◽  
Tracking Systems ◽  
Public And Private ◽  
Immunization Status ◽  
Immunization Rates ◽  
Specialized Hardware ◽  
Public And Private Cooperation ◽  
The Cost

The American Academy of Pediatrics in its role as advocate for children supports public and private cooperation in the development of immunization tracking systems (ITSs) insofar as they benefit children. All ITSs as they are developed: • Should prospectively articulate their goals and desired outcomes, including documenting immunization status and the mechanics of immunization, increasing rates of immunization, decreasing cost of immunization, and facilitating immunization opportunities; • Must accurately document each child's current immunization status; • Must preserve children's and their health care provider's right to confidentiality; • Should ensure that data will be available to health care providers 24 hours a day, 7 days a week, so that health care providers can take advantage of all opportunities to immunize; • Should ensure that data will not be used for sanctions against health care providers; • Must ensure that data input and access mechanisms enable providers to supply and access data easily, without having to purchase specialized hardware or expensive software; input and access software mechanisms need to enable all providers to supply data to and retrieve data from the ITS; • Should entitle health care providers to be reimbursed or the cost of providing data to the ITS; • Must ensure that data reflecting evidence of incomplete immunizations will not be used to deny a child access to care or eligibility for benefits by any insurance plan; • Must be studied and/or evaluated to determine their effectiveness at increasing immunization rates and decreasing costs; if such systems do not fulfill these goals, they should be eliminated; and

A Comparative Analysis of Online and Traditional Undergraduate Business Law Classes

2008 ◽  
pp. 3391-3404
Author(s):  
Daniel J. Shelley ◽  
Louis B. Swartz ◽  
Michele T. Cole
Keyword(s):  
Rural Communities ◽  
Student Satisfaction ◽  
Online Courses ◽  
Traditional Classroom ◽  
Business Law ◽  
New Paradigm ◽  
Working Adults ◽  
Student Profile ◽  
Institutions Of Higher Learning ◽  
The Cost

The trend in academia to online learning has gained momentum in the past decade, due in part to the cost of higher education, a changing student profile, lack of traditional classroom space and the recognition that distance learning has created a new paradigm of instruction. Universities wishing to maintain or expand enrollments need to be able to respond effectively to the educational needs of working adults, students in the military and residents of rural communities as well as of other countries. Online (Internet-based) course offerings constitute a creative and increasingly popular response to these challenges. As more and more institutions of higher learning offer online courses, the question arises whether they are, or can be, as effective as courses offered in the traditional classroom format. Answering the question has been the focus of several studies. Our study compared students enrolled in both online and traditional classroom versions of one business law course where all elements were the same except for the instruction format. The study found no significant difference between the two formats with regard to student satisfaction and student learning. The findings support earlier comparisons of online and traditional instruction modes

scholarly journals Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 628
Author(s):  
Elena McBeath ◽  
Jan Parker-Thornburg ◽  
Yuka Fujii ◽  
Neeraj Aryal ◽  
Chad Smith ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Sanger Sequencing ◽  
High Efficiency ◽  
Genetically Engineered ◽  
Rapid Evaluation ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetically Engineered Mice ◽  
The Cost

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

scholarly journals Building on Surface-Active Ionic Liquids for the Rescuing of the Antimalarial Drug Chloroquine

2020 ◽  
Vol 21 (15) ◽  
pp. 5334 ◽  
Author(s):  
Ana Teresa Silva ◽  
Lis Lobo ◽  
Isabel S. Oliveira ◽  
Joana Gomes ◽  
Cátia Teixeira ◽  
...  
Keyword(s):  
Ionic Liquids ◽  
Antimalarial Activity ◽  
Parent Drug ◽  
Cost Effective ◽  
Fold Increase ◽  
Pharmaceutical Ingredients ◽  
Ic50 Values ◽  
Surface Active ◽  
The Cost

Ionic liquids derived from classical antimalarials are emerging as a new approach towards the cost-effective rescuing of those drugs. Herein, we disclose novel surface-active ionic liquids derived from chloroquine and natural fatty acids whose antimalarial activity in vitro was found to be superior to that of the parent drug. The most potent ionic liquid was the laurate salt of chloroquine, which presented IC50 values of 4 and 110 nM against a chloroquine-sensitive and a chloroquine-resistant strain of Plasmodium falciparum, respectively, corresponding to an 11- and 6-fold increase in potency as compared to the reference chloroquine bisphosphate salt against the same strains. This unprecedented report opens new perspectives in both the fields of malaria chemotherapy and of surface-active ionic liquids derived from active pharmaceutical ingredients.

scholarly journals Bioeconomic modelling of grey seal predation impacts on the West of Scotland demersal fisheries

2018 ◽  
Vol 75 (4) ◽  
pp. 1374-1382 ◽  
Author(s):  
Vanessa Trijoulet ◽  
Helen Dobby ◽  
Steven J Holmes ◽  
Robin M Cook
Keyword(s):  
Status Quo ◽  
Type Ii ◽  
Grey Seal ◽  
Fishing Mortality ◽  
The West ◽  
Maximum Economic Yield ◽  
Bioeconomic Modelling ◽  
The Cost ◽  
Different Levels ◽  
Type Ii Functional Response

Abstract The role grey seals have played in the performance of fisheries is controversial and a cause of much debate between fishers and conservationists. Most studies focus on the effects of seal damage to gears or fish and on prey population abundance but little attention is given to the consequences of the latter for the fisheries. We develop a model that quantifies the economic impact of grey seal predation on the West of Scotland demersal fisheries that traditionally targeted cod, haddock and whiting. Three contrasting fishing strategy scenarios are examined to assess impacts on equilibrium fleet revenues under different levels of seal predation. These include status quo fishing mortality (SQF, steady state with constant fishing mortality), open access fishing (bioeconomic equilibrium, BE) and the maximum economic yield (MEY). In all scenarios, cod emerges as the key stock. Large whitefish trawlers are most sensitive to seal predation due to their higher cod revenues but seal impacts are minor at the aggregate fishery level. Scenarios that consider dynamic fleet behaviour also show the greatest effects of seal predation. Results are sensitive to the choice of seal foraging model where a type II functional response increases sensitivity to seal predation. The cost to the fishery for each seal is estimated.

LMP2-Cytotoxic T Lymphocyte Therapy for Relapsed EBV Positive Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3276-3276
Author(s):  
Catherine M. Bollard ◽  
Helen M. Huls ◽  
Karin C. Straathof ◽  
Stephen M. Gottschalk ◽  
Teresita Lopez ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Stable Disease ◽  
Peripheral Blood ◽  
Recombinant Adenovirus ◽  
Genetically Modified ◽  
Fold Increase ◽  
Residual Tumor ◽  
Cell Transplant ◽  
Type Ii

Abstract EBV-associated Hodgkin’s Disease (HD) and non-Hodgkins Lymphoma (NHL) show type II latency. They express the subdominant EBV antigens EBNA1, LMP1 and LMP2, which may serve as targets for immunotherapy approaches. In our previous studies, we used polyclonal EBV-specific CTL (EBV-CTL) in patients with relapsed EBV +ve HD and saw 2 complete and 1 partial response in 11 patients. Tetramer and functional analyses of EBV-CTL lines showed that small populations of T cells reactive against the tumor-associated antigen LMP2 were present in the majority of the infused lines, with some expansion in the peripheral blood following infusion. We therefore hypothesized that CTL specifically targeting type II viral latency antigens might have greater efficacy in these patients. LMP2-CTL could be generated from normal donors using dendritic cells (DC) genetically modified with a recombinant adenovirus encoding LMP2a (Ad5LMP2a) to direct the CTL response to LMP2. However, this approach required the generation of large numbers of DC to expand LMP2-CTL and was not practical in these heavily pretreated patients. We therefore modified the LMP2-CTL generation protocol to use DC for the initial stimulation, followed by stimulation with LCL that had been genetically modified to overexpress LMP2a by transduction with an Ad5f35LMP2a vector. This approach allowed us to specifically expand LMP2-CTL from patients to the numbers required for clinical use. We have generated LMP2 specific CTL lines in 10 patients with EBV+ve HD or NHL. LMP2 peptide tetramers were used for analysis of the polyclonal LMP2-CTL lines where HLA-restricted tetramers were available, and the lines recognized 2–6 (median 4) LMP2 epitopes, as determined by ELISPOT assay. A mean of 22.8% (5–42.1%) of CD8+ T cells bound these LMP2 tetramers, compared to a mean of 0.11% (0.01–0.24%) of LMP2-tetramer positive CD8+ T cells found in CTL generated with genetically unmodified LCL from the same patients. So far, 6 patients have been treated, initially receiving 2 doses of 2x107 CTL/m2 two weeks apart. All patients were off other therapies for at least 1 month prior to receiving CTL. No immediate toxicity was observed. In patients with identified LMP2-epitopes, measurement of IFNγ secretion by CD8+ T cells after stimulation with appropriate LMP2-peptides in ELISPOT assays showed a 4–25-fold increase in spot forming cells after infusions. In contrast, the frequency of CMV and superantigen-specific T cells did not increase. Four patients without radiological evidence of disease who received CTL as adjuvant therapy post stem cell transplant or chemotherapy remain well up to 9 months post CTL. Two patients with measurable disease at the time of CTL infusion had stable disease 8 weeks post CTL. They received 2 further doses of LMP2-CTL at 2x107/m2/dose. Re-evaluation was performed 8 weeks after the additional CTL infusions and one patient continues with stable disease. In the second patient, radiological review now revealed no evidence of disease, and a supraclavicular lymph node resection showed selective accumulation of LMP2-tetramer positive cells (0.3% compared to 0.01% in the peripheral blood) with scanty residual tumor cells. Immunotherapy with autologous LMP2-CTL is therefore well tolerated in patients with relapsed EBV+ve HD/NHL and infused LMP2-CTL cells can localize to the tumor and induce a clinical response.

Radiogenomics of prostate cancer: Association between qunatitative multiparametric MRI features and PTEN.

2015 ◽  
Vol 33 (7_suppl) ◽  
pp. 126-126 ◽  
Author(s):  
David James VanderWeele ◽  
Stephanie McCann ◽  
Xiaobing Fan ◽  
Tatjana Antic ◽  
Yulei Jiang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Signal Intensity ◽  
Immunohistochemical Analysis ◽  
Fold Increase ◽  
Image Features ◽  
Pten Expression ◽  
New Paradigm ◽  
Clinical Images ◽  
Mri Features ◽  
Adc Values

126 Background: Better methods are needed to assess prior to prostatectomy the risk of aggressive prostate cancer. Radiogenomics is a promising new paradigm that aims to gain molecular and genomic insights from clinical images. Loss of PTEN expression correlates with clinically aggressive disease and is associated with a 7-fold increase in the risk of prostate cancer death. Methods: From 38 patients who had undergone multi-parametric prostate MRI prior to prostatectomy, a pathologist and a radiologist simultaneously identified 45 peripheral-zone cancer regions of interest (ROIs). Histologic sections of the cancer foci underwent immunohistochemical analysis and were scored according to percent of tumor cells expressing PTEN as: negative (0-20%), mixed (20-80%), or positive (80-100%). From the MRI ROIs, the average and 10th percentile ADC values, T2-weighted signal-intensity histogram skewness, and quantitative perfusion parameters were calculated. Both dynamic perfusion two-compartment model and an empirical mathematical model (EMM) were used to fit the average contrast concentration curves within the ROIs as a function of time. Associations between the quantitative image features and PTEN expression were analyzed with Pearson's correlation coefficient (r). Results: The PTEN scores were: positive (n = 21, 47%), mixed (n = 12, 27%), and negative (n = 12, 27%). Two perfusion imaging contrast uptake parameters obtained from EMM correlated with PTEN scores (r = 0.25, p < 0.1 and r = 0.43, p < 0.01), and T2-weighted signal-intensity skewness also showed some correlation tendency (r = −0.25, p < 0.1). No correlation was seen between mean ADC and 10th percentile ADC values and PTEN score. Conclusions: This preliminary study of radiogenomics of prostate cancer suggests that fast contrast uptake of cancer on DCE-MR imaging and a T2-weighted imaging feature are potentially associated with prostate cancer PTEN expression, which warrants further studies and validation.

scholarly journals Selection for plasmid post–segregational killing depends on multiple infection: evidence for the selection of more virulent parasites through parasite–level competition

2005 ◽  
Vol 272 (1561) ◽  
pp. 403-410 ◽  
Author(s):  
T. F. Cooper ◽  
J. A. Heinemann
Keyword(s):  
Growth Rate ◽  
Growth Rates ◽  
Multiple Infection ◽  
Virulence Determinant ◽  
New Hosts ◽  
Parasite Reproduction ◽  
Selection For ◽  
The Cost ◽  
Selection Of

Is the virulence of parasites an outcome of optimized infection? Virulence has often been considered an inevitable consequence of parasite reproduction when the cost incurred by the parasite in reducing the fitness of its current host is offset by increased infection of new hosts. More recent models have focused on how competition occurring between parasites during co–infection might effect selection of virulence. For example, if co–infection was common, parasites with higher intrinsic growth rates might be selected, even at the expense of being optimally adapted to infect new hosts. If growth rate is positively correlated with virulence, then competition would select increased virulence. We tested these models using a plasmid–encoded virulence determinant. The virulence determinant did not contribute to the plasmid's reproduction within or between hosts. Despite this, virulent plasmids were more successful than avirulent derivatives during selection in an environment allowing within–host competition. To explain these findings we propose and test a model in which virulent parasites are selected by reducing the reproduction of competitors.

scholarly journals Technology of timber harvesting with preservation of undergrowth

2015 ◽  
Vol 5 (1) ◽  
pp. 136-143
Author(s):  
Дербин ◽  
Vasiliy Derbin ◽  
Дербин ◽  
Mikhail Derbin
Keyword(s):  
New Technologies ◽  
Unit Area ◽  
Fold Increase ◽  
Timber Harvesting ◽  
Load Factor ◽  
Technological Operation ◽  
Forest Road ◽  
Surface Cover ◽  
Full Load ◽  
The Cost

In recent years, requirements of certification bodies and forestry authorities for developed to felling in terms of the number of undergrowth per unit area are increased. To save the undergrowth and reducing the area of damage to the surface cover cutting areas it is most advisable to use wide-feller bunchers, for example, ЛП-19, МЛ-119, Timberjack 850, John Deere 753J, John Deere 759J, etc.. Number of undergrowth largely depends on the development plan of cutting areas development. Known technological schemes are reviewed and analyzed. When hauling packs of trees at one spur track of forest road feller-buncher moves with the rapid moves, which reduce the efficiency of the development of cutting areas as a whole. When hauling trees packs at two spur track of logging roads two fold increase in the length of spur tracks is needed, which greatly increases the effort required to perform logging operations and the cost of production? New technologies for development of cutting areas are presented. In this case, traffic schemes of track feller buncher and skidder are the same as the known technology of timber harvesting without undergrowth preservation. Saving undergrowth is provided by changing the sequence of execution of elements of technological operation for forming full-load for bundles of trees for skidding. Upon developing cutting areas on the proposed technologies processes of formation of full-load skidding bundles of trees is accompanied by a rise of trees above the ground from packs formed by feller bunchers, and transfer them to skidding trails. Performance of skidder is defined. When comparing all the considered schemes to develop cutting areas several factors must be considered: load factor of skidder, distance of idling, duration of stacking trees in the forming device, possible change in the rate of tractor stroke due to the different download.

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