Search

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.

CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two Higher Eukaryotes and Prokaryotes Nucleotide (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.

CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming multi-gene expression, but poor predictability of guide RNA folding can disrupt expression control. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA folding rate into the active structure (rS = 0.8). This parameter also enables forward design of scRNAs, allowing us to design a system of three synthetic CRISPRa promoters that can orthogonally activate (>35-fold) expression of chosen outputs. Through combinatorial activation tuning, we profile a three-dimensional design space expressing two different biosynthetic pathways, demonstrating variable production of pteridine and human milk oligosaccharide products. This RNA design approach aids combinatorial optimization of metabolic pathways and may accelerate routine design of effective multi-gene regulation programs in bacterial hosts.

Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).

Introduction: Thyroid nodules are extremely common being detected by ultrasonography in up to 67% of the population, with current surgical tenet maintaining that lobectomy is required for large symptomatic benign nodules or autonomously functionally nodules resulting in a risk of hypothyroidism or recurrent laryngeal nerve injury even in high volume centres. The introduction of radiofrequency ablation (RFA) has allowed thermal ablation of both benign and autonomously functioning thyroid nodules with minimal morbidity. The moving shot technique is the most well-established technique in performing RFA of thyroid nodules, and has proven to be safe, efficacious, accurate and successful amongst experienced clinicians. The purpose of this article to propose the use of a novel guide when performing RFA of thyroid nodules in clinical practice utilizing the moving shot technique. Methods: The technique proposed of RFA involves the use of a 10MHz linear ultrasound probe attached to an 18G guide which provides robust in line visualisation of a 7cm or 10cm radiofrequency probe tip (STARmed, Seoul, Korea) utilizing the trans isthmic moving shot technique. A geometric analysis of the guide has been illustrated diagrammatically. Results: The use of an 18G radiofrequency probe guide (CIVCO Infiniti Plus™ Needle Guide) maintains in line visualisation of the radiofrequency probe over a cross-sectional area up to 28cm2, facilitating efficient and complete ablation of conceptual subunits during RFA of thyroid nodules. Discussion: Radiofrequency ablation of thyroid nodules can be performed safely and effectively using the novel radiofrequency probe guide proposed which we believe potentially improves both accuracy and overall efficiency, along with operator confidence in maintaining visualisation of the probe tip, and hence we believe provides a valuable addition to the armamentarium of clinicians wishing to embark on performing RFA of thyroid nodules. [ABSTRACT FROM AUTHOR]

Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.

Genetic logic gates can be employed in metabolic engineering and synthetic biology to regulate gene expression based on diverse inputs. Design of tunable genetic logic gates with versatile dynamic performance is essential for expanding the usability of these toolsets. Here, using the p-coumaric acid biosensor system as a proof-of-concept, we initially investigated the parameters influencing the buffer (BUF) genetic logic gates. Subsequently, integrating binding sequences from the p-coumaric acid biosensor system and tetR or lacI regulation systems into a constitutive promoter yielded AND genetic logic gates. Additionally, characterized antisense RNAs (asRNAs) or single guide RNAs (sgRNAs) with various repression efficiencies were combined with BUF gates to construct a suite of p-coumaric acid-triggered NOT genetic logic gates. Finally, the designed BUF and NOT gates were combined to construct bifunctional genetic circuits that were subjected to orthogonality evaluation. The genetic logic gates established in this study can serve as valuable tools in future applications of metabolic engineering and synthetic biology.

CRISPR-Cas9 genome editing has promising therapeutic potential for genetic diseases and cancers, but safety could be a concern. Here we use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing and in comparison to four parental cell lines. In addition to the previously reported large structural variants at on-target sites, we identify heretofore unexpected large chromosomal deletions (91.2 and 136 Kb) at atypical non-homologous off-target sites without sequence similarity to the sgRNA in two edited lines. The observed large structural variants induced by CRISPR-Cas9 editing in dividing cells may result in pathogenic consequences and thus limit the usefulness of the CRISPR-Cas9 editing system for disease modeling and gene therapy. In this work, our whole genomic analysis may provide a valuable strategy to ensure genome integrity after genomic editing to minimize the risk of unintended effects in research and clinical applications.

Background & Aims: Mental health literacy training programs increase knowledge about mental health and mental illness among teenagers, which ultimately reduces the stigma associated with mental illness and increases help-seeking behavior. This study aims to investigate school-based mental health literacy programs in high school students. Materials & Methods: This study is a scoping review aimed at examining school-based mental health literacy programs in high school students. Studies published from the first year of the article’s publication related to the topic until April 10, 2024, were reviewed. Databases including Scopus, Web of Science, PubMed, and ProQuest, as well as three Iranian scientific databases — Scientific Information Database (SID), Islamic World Science Citation Database (ISC), and Magiran for Persian studies, were searched using keywords of school-based mental health literacy, school-based mental hygiene literacy, school-based mental health, school-based mental hygiene, intervention, program, and curriculum. Results: Considering the inclusion and exclusion criteria, the studies will be examined and analyzed, and the information pertaining to these studies will be reported in accordance with the type of school-based mental health literacy training program, the training framework of the program, the program's objectives, and the findings. Conclusion: In student mental health literacy programs, if incorporating mental health education into school curricula is the ultimate goal, it makes sense for teachers to provide a classroom-based curriculum as they are likely to be the ones putting this into practice in the real world.

In the modern world, contacts between cultures tend to strengthen, even during and after military conflicts. After France had been liberated by the US Army during World War II, US soldiers started getting acquainted with French culture. This article introduces an analysis of American guidebooks published specifically for American soldiers who served in France during World War II. The research objective was to identify the information that the guidebooks represented as necessary for an average US military person to make their stay in the country more comfortable, e.g., culture, entertainment, recreation, etc. The guidebooks told their American readers about the history of France and its current state, as well as about the relationship between the French, Americans, and Germans during the world wars. The guides aimed at facilitating the adaptation of American military personnel to French environment. Initially, the guidebooks attempted to introduce American soldiers to the basic knowledge about France, but gradually they started to publish advice on practical issues that Americans faced in France. The guidebooks were written by prominent representatives of American culture who worked for the armed forces during World War II. All in all, these guidebooks enhanced American interest in French culture.

Abstract Background Patient-specific aiming devices (PSAD) may improve precision and accuracy of glenoid component positioning in total shoulder arthroplasty, especially in degenerative glenoids. The aim of this study was to compare precision and accuracy of guide wire positioning into different glenoid models using a PSAD versus a standard guide. Methods Three experienced shoulder surgeons inserted 2.5 mm K-wires into polyurethane cast glenoid models of type Walch A, B and C (in total 180 models). Every surgeon placed guide wires into 10 glenoids of each type with a standard guide by DePuy Synthes in group (I) and with a PSAD in group (II). Deviation from planned version, inclination and entry point was measured, as well as investigation of a possible learning curve. Results Maximal deviation in version in B- and C-glenoids in (I) was 20.3° versus 4.8° in (II) (p

Artificial intelligence (AI) has significant potential to transform healthcare and improve patient care. However, successful development and integration of AI models requires careful consideration of study designs and sample size calculations for development and validation of models, publishing standards, prototype development for translation and collaboration with stakeholders. As the field is relatively new and rapidly evolving there is a lack of guidance and agreement on best practices for most of these steps. We engaged stakeholders in the form of clinicians, researchers from academia and industry, and data scientists to discuss various aspects of the translational pipeline and identified the challenges researchers in the field face and potential solutions to them. In this viewpoint, we present the summary of our discussions as a brief guide on the process of developing AI-based applications for healthcare using medical images. We organized the entire process into six major themes (i.e., The gaps AI can fill in healthcare, Development of AI models for healthcare: practical and important things to consider, Good practices for validation of AI models for healthcare: study designs and sample size calculation, Points to consider when publishing AI models, Translation towards products, Challenges and potential solutions from a technical perspective) and presented important points as a rule of thumb. We conclude that successful integration of AI in healthcare requires a collaborative approach, rigorous validation, adherence to best practices as described and cited, and consideration of technical aspects.

The piece "Da pacem Domine" (Give us peace, O Lord), by Arvo Part was inspired by a Gregorian antiphon from the 9th century. Written in the original tintinnabuli style, the piece uses numerous compositional techniques belonging to the early Renaissance period called Cantus firmus, parallel organum, faux bourdon, Landini cadence or anticipations, but also and even a modern way of vocal scoring: the "pointillistic polyphony". The work is divided into four sections, corresponding to the four verses of the prayer and the gravitational centre of the music is D. To impart expressive meaning to the musical performance, the interprets, both the conductor or singers can consider the following elements: homogeneity in individual voices and ensemble performance, different singing modalities (legato/non-legato) alternating between moments of cadence and the rest of the piece, the technique of attack and sound placement, finding and maintaining an appropriate inner state reflecting the ideas of the text and the general character of the piece. [ABSTRACT FROM AUTHOR]

Background: Patient-specific aiming devices (PSAD) may improve precision and accuracy of glenoid component positioning in total shoulder arthroplasty, especially in degenerative glenoids. The aim of this study was to compare precision and accuracy of guide wire positioning into different glenoid models using a PSAD versus a standard guide. Methods: Three experienced shoulder surgeons inserted 2.5 mm K-wires into polyurethane cast glenoid models of type Walch A, B and C (in total 180 models). Every surgeon placed guide wires into 10 glenoids of each type with a standard guide by DePuy Synthes in group (I) and with a PSAD in group (II). Deviation from planned version, inclination and entry point was measured, as well as investigation of a possible learning curve. Results: Maximal deviation in version in B- and C-glenoids in (I) was 20.3° versus 4.8° in (II) (p < 0.001) and in inclination was 20.0° in (I) versus 3.7° in (II) (p < 0.001). For B-glenoid, more than 50% of the guide wires in (I) had a version deviation between 11.9° and 20.3° compared to ≤ 2.2° in (II) (p < 0.001). 50% of B- and C-glenoids in (I) showed a median inclination deviation of 4.6° (0.0°-20.0°; p < 0.001) versus 1.8° (0.0°-4.0°; p < 0.001) in (II). Deviation from the entry point was always less than 5.0 mm when using PSAD compared to a maximum of 7.7 mm with the standard guide and was most pronounced in type C (p < 0.001). Conclusion: PSAD enhance precision and accuracy of guide wire placement particularly for deformed B and C type glenoids compared to a standard guide in vitro. There was no learning curve for PSAD. However, findings of this study cannot be directly translated to the clinical reality and require further corroboration. [ABSTRACT FROM AUTHOR]

Objectives: The peer review process is critical to maintaining quality, reliability, novelty, and innovation in the scientific literature. However, the teaching of scientific peer review is rarely a component of formal scientific or clinical training, and even the most experienced peer reviewers express interest in continuing education. The objective of this review article is to summarize the collective perspectives of experienced journal editors about how to be a good reviewer in a step-by-step guide that can serve as a resource for the performance of peer review of a scientific manuscript. Methods: This is a narrative review. Results: A review of the history and an overview of the modern-day peer review process are provided with attention to the role played by the reviewer, including important reasons for involvement in scientific peer review. The general components of a scientific peer review are described, and a model for how to structure a peer review report is provided. These concepts are also summarized in a reviewer checklist that can be used in real-time to develop and double-check one's reviewer report before submitting it. Conclusions: Peer review is a critically important service for maintaining quality in the scientific literature. Peer review of a scientific manuscript and the associated reviewer's report should assess specific details related to the accuracy, validity, novelty, and interpretation of a study's results. We hope that this article will serve as a resource and guide for reviewers of all levels of experience in the performance of peer review of a scientific manuscript. [ABSTRACT FROM AUTHOR]

Background and Aim: The study's purpose is to improve the quality of care for both local residents and travelers in the Kurnool district by identifying and addressing gaps and barriers. The main objective of the research was to assess the knowledge, attitudes, and practical implementation of travel medicine among primary health-care physicians working in the Kurnool district. Materials and Methods: The research utilized a descriptive cross-sectional study design. A total of 397 primary care doctors in the Kurnool area participated. Data were collected through a self-administered questionnaire, which covered sociodemographic factors, travel medicine knowledge, attitudes, and practices. Results: The study revealed that a significant portion (around 79.5%) of the participants reported providing travel health advice at some point. The primary source of information on travel medicine for most participants (66%) was the Ministry of Health's travel guides. However, the study identified a knowledge gap, as 11.4% of individuals were unaware of the origin of typhoid infection. The most common health issues reported during posttrip consultations were diarrhea and respiratory infections. Conclusion: The study's findings suggested that the general practitioners in the Kurnool district had insufficient knowledge, attitudes, and practices concerning travel medicine. This underscores the need for additional education and training in travel medicine to improve traveler health care and reduce the risk of preventable diseases. [ABSTRACT FROM AUTHOR]

In Trypanosoma brucei, the editosome, composed of RNA-editing substrate-binding complex (RESC) and RNA-editing catalytic complex (RECC), orchestrates guide RNA (gRNA)-programmed editing to recode cryptic mitochondrial transcripts into messenger RNAs (mRNAs). The mechanism of information transfer from gRNA to mRNA is unclear owing to a lack of high-resolution structures for these complexes. With cryo-electron microscopy and functional studies, we have captured gRNA-stabilizing RESC-A and gRNA-mRNA-binding RESC-B and RESC-C particles. RESC-A sequesters gRNA termini, thus promoting hairpin formation and blocking mRNA access. The conversion of RESC-A into RESC-B or -C unfolds gRNA and allows mRNA selection. The ensuing gRNA-mRNA duplex protrudes from RESC-B, likely exposing editing sites to RECC-catalyzed cleavage, uridine insertion or deletion, and ligation. Our work reveals a remodeling event facilitating gRNA-mRNA hybridization and assembly of a macromolecular substrate for the editosome's catalytic modality.

While a number of methods exist to investigate CRISPR off-target (OT) editing, few have been compared head-to-head in primary cells after clinically relevant editing processes. Therefore, we compared in silico tools (COSMID, CCTop, and Cas-OFFinder) and empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) after ex vivo hematopoietic stem and progenitor cell (HSPC) editing. We performed editing using 11 different gRNAs complexed with Cas9 protein (high-fidelity [HiFi] or wild-type versions), then performed targeted next-generation sequencing of nominated OT sites identified by in silico and empirical methods. We identified an average of less than one OT site per guide RNA (gRNA) and all OT sites generated using HiFi Cas9 and a 20-nt gRNA were identified by all OT detection methods with the exception of SITE-seq. This resulted in high sensitivity for the majority of OT nomination tools and COSMID, DISCOVER-Seq, and GUIDE-Seq attained the highest positive predictive value (PPV). We found that empirical methods did not identify OT sites that were not also identified by bioinformatic methods. This study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity and PPV, thereby enabling more efficient identification of potential OT sites without compromising a thorough examination for any given gRNA.

The role of teacher as a guide is very significant in teaching, especially to equip students to know God through Christian education. Teachers guide the students to understand knowledge, attitudes, skills, and spirituality based on Biblical principles. But in reality, teachers do not teach the Bible principles in learning, teachers do not guide the students, and have lack of knowledge of the Bible. Teachers should have understood the significance of guiding students based on the Bible. Hence, the purpose of writing this article is to present the significant role of teachers as a guide through the theological study based on Ephesians 4:11-16 for the context of Christian education with literature study as the research method. Teachers must guide students based on the truth of God's Word, through imitating Jesus as the Great Shepherd. Christian teachers’ role as guide do not only stop at knowledge in academic learning, but to be an impact for students to be a part of service to God and to build others up as members of the body of Christ. Christian teachers must guide students to achieve, namely: the unity of the faith in Christ as God and Savior in true knowledge, thorough maturity, and growth in accordance with the fullness of Christ and to ensure to be not tossed around by the waves of false teaching that ignore The Lord. This article concludes that the teacher's role as guide is one form for equipping students to grow more mature in Christ. The author suggests that Christian teachers understand that teachers are God's calling to reveal God's truth to students so that their lives base on Bible teaching, and Christian teachers must understand the students' needs to know God and grow spiritually to become more like Christ. BAHASA INDONESIA ABSTRACT: Peran guru sebagai pembimbing sangat penting dalam pengajaran, terutama untuk memperlengkapi murid-murid mengenal Tuhan melalui pendidikan Kristen. Guru membimbing murid untuk memahami pengetahuan, sikap, keterampilan, dan kerohanian berdasarkan prinsip-prinsip Alkitab. Namun pada kenyataannya, guru tidak mengajarkan prinsip-prinsip Alkitab dalam pembelajaran, guru tidak membimbing siswa, dan memiliki pengetahuan yang kurang tentang Alkitab. Seharusnya para guru memahami pentingnya membimbing murid berdasarkan Alkitab. Oleh karena itu, tujuan dari penulisan artikel ini adalah untuk memaparkan peran penting guru sebagai pembimbing melalui kajian teologis berdasarkan Efesus 4:11-16 dalam konteks pendidikan Kristen dengan metode studi literatur. Guru harus membimbing siswa berdasarkan kebenaran Firman Tuhan, dengan meneladani Yesus sebagai Gembala Agung. Peran guru Kristen sebagai pembimbing tidak hanya berhenti pada pengetahuan dalam pembelajaran akademis, tetapi menjadi dampak bagi siswa untuk menjadi bagian dari pelayanan kepada Tuhan dan membangun orang lain sebagai anggota tubuh Kristus. Guru Kristen harus membimbing murid-muridnya untuk mencapai, yaitu: kesatuan iman kepada Kristus sebagai Tuhan dan Juruselamat dalam pengetahuan yang benar, kedewasaan yang menyeluruh, dan pertumbuhan yang sesuai dengan kepenuhan Kristus serta memastikan agar tidak terombang-ambing oleh gelombang pengajaran palsu yang mengabaikan Tuhan. Artikel ini menyimpulkan bahwa peran guru sebagai pembimbing adalah salah satu bentuk untuk memperlengkapi murid-muridnya agar bertumbuh semakin dewasa di dalam Kristus. Penulis menyarankan agar para guru Kristen memahami bahwa guru adalah panggilan Tuhan untuk menyatakan kebenaran Tuhan kepada murid-muridnya agar hidup mereka didasarkan pada pengajaran Alkitab, dan guru Kristen harus memahami kebutuhan murid-muridnya untuk mengenal Tuhan dan bertumbuh secara rohani agar semakin serupa dengan Kristus.

Abstract Objective Evaluation of the accuracy and effectiveness of 3D printed guides to assist femoral tunnel preparation in individualised reconstruction of the anterior cruciate ligament. Methods Sixty patients who attended the Affiliated Hospital of Binzhou Medical College for autologous hamstring single bundle reconstruction of the anterior cruciate ligament from October 2018 to October 2020 were selected and randomly divided into two groups, including 31 cases in the 3D printing group (14 males and 17 females, mean age 41.94 ± 10.15 years) and 29 cases in the control group (13 males and 16 females, mean age 37.76 ± 10.34 years). Patients in both groups were assessed for intraoperative femoral tunnel accuracy, the number of intraoperative positioning and the time taken to prepare the femoral tunnel, the length of the anteromedial approach incision, the pre-planned bone tunnel length and intraoperative bone tunnel length in the 3D printed group, IKDC score and Lysholm score preoperatively and at 3, 6 and 12 months postoperatively, the Lachman、pivot-shift test preoperatively and at 6 months postoperatively, gait analysis to assess internal and external rotation in flexion of the knee at 12 months postoperatively and postoperative complications in both groups. Results There was no statistical difference in functional knee scores and anteromedial approach incision length between the 3D printed and control groups (p > 0.05), while there was a statistical difference in the accuracy of tunnel positioning, the time taken to prepare the femoral bone tunnel and the degree of external rotation of the knee in flexion between the two groups (p 0.05). Complications: One case in the 3D printing group developed intermuscular vein thrombosis in the affected lower limb after surgery, which disappeared after treatment, while three cases in the control group developed intermuscular vein thrombosis in the affected lower limb. No complications such as bone tunnel rupture, deep vein thrombosis in the lower limb and infection occurred in either group. Conclusion 3D printed guides assisted with individualized ACL reconstruction may improve the accuracy of femoral tunnel positioning, which is safe and effective, while reducing the operative time and the number of intraoperative positioning, without increasing the length of incision, and may obtain higher functional scores and rotational stability of the knee joint, which is in line with the concept of individualized ACL reconstruction.

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

BackgroundZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create 'knockout' models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies.MethodsTo systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression.ResultsWe compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (

Understanding the numerous roles that colouration serves in the natural world has remained a central focus in many evolutionary and ecological studies. However, to accurately characterise and then compare colours or patterns among individuals or species has been historically challenging. In recent years, there have been a myriad of new resources developed that allow researchers to characterise biological colours and patterns, specifically from digital imagery. However, each resource has its own strengths and weaknesses, answers a specific question and requires a detailed understanding of how it functions to be used properly. These nuances can make navigating this emerging field rather difficult. Herein, we evaluate several new techniques for analysing biological colouration, with a specific focus on digital images. First, we introduce fundamental background knowledge about light and perception to be considered when designing and implementing a study of colouration. We then show how numerous modifications can be made to images to ensure consistent formatting prior to analysis. After, we describe many of the new image analysis approaches and their respective functions, highlighting the type of research questions that they can address. We demonstrate how these various techniques can be brought together to examine novel research questions and test specific hypotheses. Finally, we outline potential future directions in colour pattern studies. Our goal is to provide a starting point and pathway for researchers wanting to study biological colour patterns from digital imagery. [ABSTRACT FROM AUTHOR]

Reducing building energy use, a linchpin of climate change mitigation, is a daunting challenge across the world. Gaining increasing attention, retro-commissioning (RCx) is a systematic process that can improve building energy performance. Using a techno-legal lens to review statutes and guides on RCx, this study reveals that in China, a national standard on building commissioning has taken effect yet RCx statutes are hitherto not found. The United States has RCx statutes enacted over 14 states; scrutinizing the statutes of five cities unveils similarities and differences in their scope of control and compliance requirements. In the absence of a specific RCx statute, the guide of Hong Kong, China provides detailed guidance for energy saving practices. While these findings can serve as reference for other places planning to formulate laws or guidance on RCx, the need to further study the effectiveness of mandating RCx for reducing building energy use is highlighted. A conceptual analysis of cost variation with statutory control, which could help policymakers consider from an economic perspective whether or to what extent statutory RCx requirements should be imposed, is also illustrated. This not only contributes insights to the pursuit of an optimal balance between statutory control and voluntary action for energy reduction but also adds to the debate on building energy policies. [ABSTRACT FROM AUTHOR]

Objective: Evaluation of the accuracy and effectiveness of 3D printed guides to assist femoral tunnel preparation in individualised reconstruction of the anterior cruciate ligament. Methods: Sixty patients who attended the Affiliated Hospital of Binzhou Medical College for autologous hamstring single bundle reconstruction of the anterior cruciate ligament from October 2018 to October 2020 were selected and randomly divided into two groups, including 31 cases in the 3D printing group (14 males and 17 females, mean age 41.94 ± 10.15 years) and 29 cases in the control group (13 males and 16 females, mean age 37.76 ± 10.34 years). Patients in both groups were assessed for intraoperative femoral tunnel accuracy, the number of intraoperative positioning and the time taken to prepare the femoral tunnel, the length of the anteromedial approach incision, the pre-planned bone tunnel length and intraoperative bone tunnel length in the 3D printed group, IKDC score and Lysholm score preoperatively and at 3, 6 and 12 months postoperatively, the Lachman、pivot-shift test preoperatively and at 6 months postoperatively, gait analysis to assess internal and external rotation in flexion of the knee at 12 months postoperatively and postoperative complications in both groups. Results: There was no statistical difference in functional knee scores and anteromedial approach incision length between the 3D printed and control groups (p > 0.05), while there was a statistical difference in the accuracy of tunnel positioning, the time taken to prepare the femoral bone tunnel and the degree of external rotation of the knee in flexion between the two groups (p < 0.05). There was no statistical difference between the preoperative planning of the bone tunnel length and the intraoperative bone tunnel length (p > 0.05). Complications: One case in the 3D printing group developed intermuscular vein thrombosis in the affected lower limb after surgery, which disappeared after treatment, while three cases in the control group developed intermuscular vein thrombosis in the affected lower limb. No complications such as bone tunnel rupture, deep vein thrombosis in the lower limb and infection occurred in either group. Conclusion: 3D printed guides assisted with individualized ACL reconstruction may improve the accuracy of femoral tunnel positioning, which is safe and effective, while reducing the operative time and the number of intraoperative positioning, without increasing the length of incision, and may obtain higher functional scores and rotational stability of the knee joint, which is in line with the concept of individualized ACL reconstruction. [ABSTRACT FROM AUTHOR]

Lateral lumbar interbody fusion (LLIF) is a popular technique as it allows for the placement of a large interbody implant through a retroperitoneal, transpsoas working corridor. Historically, the interbody is placed with the patient in lateral decubitus and then repositioned to prone for the posterior instrumentation. While this has been an effective and successful technique, removing the interoperative flip would improve the efficiency of these cases. This has led to modified LLIF approaches including single-position prone LLIF (pLLIF). This modification has shown to be an efficient and powerful technique; however, learning to navigate the LLIF approach in the prone position has its own challenges. The purpose of this article is to provide a detailed description of our pLLIF technique while simultaneously introducing surgical tips to overcome the challenges of the approach and optimize the implantation of the interbody device. Novel Techniques & Technology. [ABSTRACT FROM AUTHOR]

IntroductionThyroid nodules are extremely common being detected by ultrasonography in up to 67% of the population, with current surgical tenet maintaining that lobectomy is required for large symptomatic benign nodules or autonomously functionally nodules resulting in a risk of hypothyroidism or recurrent laryngeal nerve injury even in high volume centres. The introduction of radiofrequency ablation (RFA) has allowed thermal ablation of both benign and autonomously functioning thyroid nodules with minimal morbidity. The moving shot technique is the most well-established technique in performing RFA of thyroid nodules, and has proven to be safe, efficacious, accurate and successful amongst experienced clinicians. The purpose of this article to propose the use of a novel guide when performing RFA of thyroid nodules in clinical practice utilizing the moving shot technique.MethodsThe technique proposed of RFA involves the use of a 10MHz linear ultrasound probe attached to an 18G guide which provides robust in line visualisation of a 7cm or 10cm radiofrequency probe tip (STARmed, Seoul, Korea) utilizing the trans isthmic moving shot technique. A geometric analysis of the guide has been illustrated diagrammatically.ResultsThe use of an 18G radiofrequency probe guide (CIVCO Infiniti Plus™ Needle Guide) maintains in line visualisation of the radiofrequency probe over a cross-sectional area up to 28cm2, facilitating efficient and complete ablation of conceptual subunits during RFA of thyroid nodules.DiscussionRadiofrequency ablation of thyroid nodules can be performed safely and effectively using the novel radiofrequency probe guide proposed which we believe potentially improves both accuracy and overall efficiency, along with operator confidence in maintaining visualisation of the probe tip, and hence we believe provides a valuable addition to the armamentarium of clinicians wishing to embark on performing RFA of thyroid nodules.

Abstract Objectives The peer review process is critical to maintaining quality, reliability, novelty, and innovation in the scientific literature. However, the teaching of scientific peer review is rarely a component of formal scientific or clinical training, and even the most experienced peer reviewers express interest in continuing education. The objective of this review article is to summarize the collective perspectives of experienced journal editors about how to be a good reviewer in a step‐by‐step guide that can serve as a resource for the performance of peer review of a scientific manuscript. Methods This is a narrative review. Results A review of the history and an overview of the modern‐day peer review process are provided with attention to the role played by the reviewer, including important reasons for involvement in scientific peer review. The general components of a scientific peer review are described, and a model for how to structure a peer review report is provided. These concepts are also summarized in a reviewer checklist that can be used in real‐time to develop and double‐check one's reviewer report before submitting it. Conclusions Peer review is a critically important service for maintaining quality in the scientific literature. Peer review of a scientific manuscript and the associated reviewer's report should assess specific details related to the accuracy, validity, novelty, and interpretation of a study's results. We hope that this article will serve as a resource and guide for reviewers of all levels of experience in the performance of peer review of a scientific manuscript.

Particulate nitrate, a key component of fine particles, forms through the intricate gas-to-particle conversion process. This process is regulated by the gas-to-particle conversion coefficient of nitrate (ε(NO3−)). The mechanism between ε(NO3−) and its drivers is highly complex and nonlinear, and can be characterized by machine learning methods. However, conventional machine learning often yields results that lack clear physical meaning and may even contradict established physical/chemical mechanisms due to the influence of ambient factors. It urgently needs an alternative approach that possesses transparent physical interpretations and provides deeper insights into the impact of ε(NO3−). Here we introduce a supervised machine learning approach—the multilevel nested random forest guided by theory approaches. Our approach robustly identifies NH4+, SO42−, and temperature as pivotal drivers for ε(NO3−). Notably, substantial disparities exist between the outcomes of traditional random forest analysis and the anticipated actual results. Furthermore, our approach underscores the significance of NH4+ during both daytime (30%) and nighttime (40%) periods, while appropriately downplaying the influence of some less relevant drivers in comparison to conventional random forest analysis. This research underscores the transformative potential of integrating domain knowledge with machine learning in atmospheric studies.

Each of the three similar RNA Editing Catalytic Complexes (RECCs) that perform gRNA-directed uridine insertion and deletion during Trypanosoma brucei mitochondrial (mt) mRNA editing has a distinct endonuclease activity that requires two related RNase III proteins, with only one competent for catalysis. We identified multiple loss-of-function mutations in the RNase III and other motifs of the non-catalytic KREPB6, KREPB7, and KREPB8 components by random mutagenesis and screening. These mutations had various effects on growth, editing, and both the abundances and RECC associations of these RNase III protein pairs in bloodstream form (BF) and procyclic form (PF) cells. Protein structure modelling predicted that the Zinc Finger (ZnF) of each paired RNase III protein contacts RNA positioned at the heterodimeric active site which is flanked by helices of a novel RNase III-Associated Motif (RAM). The results indicate that the protein domains of the non-catalytic subunits function together in RECC integrity, substrate binding, and editing site recognition during the multistep RNA editing process. Additionally, several mutants display distinct functional consequences in different life cycle stages. These results highlight the complementary roles of protein pairs and three RECCs within the complicated T. brucei mRNA editing machinery that matures mt mRNAs differentially between developmental stages.

Providing guidance to learners navigating a game-based learning environment requires walking a fine line between encouraging progression toward learning goals without disrupting playful engagement in the game. In this paper, we present a scaffolding guidebook developed for tutors to provide guidance in game-based learning environments that encourages exploration of the problem space and solving puzzles without disrupting engagement. Scaffolding strategies were coded and categorized from Baba is You gameplay recordings of 13 middle school and 12 undergraduate students and then situated based on guiding principles from relevant literature into a scaffolding guide. Here, we describe this guidebook and its development, which could provide educators with important tools that can help their students progress through game-based learning environments without interfering with engagement.

Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.

Background and Aim: The study’s purpose is to improve the quality of care for both local residents and travelers in the Kurnool district by identifying and addressing gaps and barriers. The main objective of the research was to assess the knowledge, attitudes, and practical implementation of travel medicine among primary health-care physicians working in the Kurnool district. Materials and Methods: The research utilized a descriptive cross-sectional study design. A total of 397 primary care doctors in the Kurnool area participated. Data were collected through a self-administered questionnaire, which covered sociodemographic factors, travel medicine knowledge, attitudes, and practices. Results: The study revealed that a significant portion (around 79.5%) of the participants reported providing travel health advice at some point. The primary source of information on travel medicine for most participants (66%) was the Ministry of Health’s travel guides. However, the study identified a knowledge gap, as 11.4% of individuals were unaware of the origin of typhoid infection. The most common health issues reported during posttrip consultations were diarrhea and respiratory infections. Conclusion: The study’s findings suggested that the general practitioners in the Kurnool district had insufficient knowledge, attitudes, and practices concerning travel medicine. This underscores the need for additional education and training in travel medicine to improve traveler health care and reduce the risk of preventable diseases.

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remain low using current guide RNA designs. In this study, we engineered circular ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.

Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.

Copyright of Przegląd Pedagogiczny is the property of Kazimierza Wielki University in Bydgoszcz and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Understanding the numerous roles that colouration serves in the natural world has remained a central focus in many evolutionary and ecological studies. However, to accurately characterise and then compare colours or patterns among individuals or species has been historically challenging. In recent years, there have been a myriad of new resources developed that allow researchers to characterise biological colours and patterns, specifically from digital imagery. However, each resource has its own strengths and weaknesses, answers a specific question and requires a detailed understanding of how it functions to be used properly. These nuances can make navigating this emerging field rather difficult. Herein, we evaluate several new techniques for analysing biological colouration, with a specific focus on digital images. First, we introduce fundamental background knowledge about light and perception to be considered when designing and implementing a study of colouration. We then show how numerous modifications can be made to images to ensure consistent formatting prior to analysis. After, we describe many of the new image analysis approaches and their respective functions, highlighting the type of research questions that they can address. We demonstrate how these various techniques can be brought together to examine novel research questions and test specific hypotheses. Finally, we outline potential future directions in colour pattern studies. Our goal is to provide a starting point and pathway for researchers wanting to study biological colour patterns from digital imagery.

Relative cerebral blood volume (rCBV) derived from dynamic susceptibility contrast (DSC) perfusion MR imaging (pMRI) has been shown to be a robust marker of neuroradiological tumor burden. Recent consensus recommendations in pMRI acquisition strategies have provided a pathway for pMRI inclusion in diverse patient care centers, regardless of size or experience. However, even with proper implementation and execution of the DSC-MRI protocol, issues will arise that many centers may not easily recognize or be aware of. Furthermore, missed pMRI issues are not always apparent in the resulting rCBV images, potentiating inaccurate or missed radiological diagnoses. Therefore, we gathered from our database of DSC-MRI datasets, true-to-life examples showcasing the breakdowns in acquisition, postprocessing, and interpretation, along with appropriate mitigation strategies when possible. The pMRI issues addressed include those related to image acquisition and postprocessing with a focus on contrast agent administration, timing, and rate, signal-to-noise quality, and susceptibility artifact. The goal of this work is to provide guidance to minimize and recognize pMRI issues to ensure that only quality data is interpreted.

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.

A compact protein with a size of

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.

Abstract Background Few training opportunities or resources for non-expert implementers focus on the “practice” as opposed to the “science” of knowledge translation (KT). As a guide for novice implementers, we present an open-access, fillable workbook combining KT theories, models, and frameworks (TMFs) that are commonly used to support the implementation of evidence-based practices. We describe the process of creating and operationalizing our workbook. Methods Our team has supported more than 1000 KT projects and 300 teams globally to implement evidence-based interventions. Our stakeholders have consistently highlighted their need for guidance on how to operationalize various KT TMFs to support novice implementers in “practising” KT. In direct response to these requests, we created a pragmatic, fillable KT workbook. The workbook was designed by KT scientists and experts in the fields of adult education, graphic design, and usability and was piloted with novice implementers. It is rooted in an integrated KT approach and applies an intersectionality lens, which prompts implementers to consider user needs in the design of implementation efforts. Results The workbook is framed according to the knowledge-to-action model and operationalizes each stage of the model using appropriate theories or frameworks. This approach removes guesswork in selecting appropriate TMFs to support implementation efforts. Implementers are prompted to complete fillable worksheets that are informed by the Theoretical Domains Framework, the Consolidated Framework for Implementation Research, the Behaviour Change Wheel, the Effective Practice and Organization of Care framework, Proctor’s operationalization framework, the Durlak and DuPre process indicators, and the Reach, Effectiveness, Adoption, Implementation and Maintenance (RE-AIM) framework. As they complete the worksheets, users are guided to apply theoretically rooted approaches in planning the implementation and evaluation of their evidence-based practice. Conclusions This workbook aims to support non-expert implementers to use KT TMFs to select and operationalize implementation strategies to facilitate the implementation of evidence-based practices. It provides an accessible option for novice implementers who wish to use KT methods to guide their work.

As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.