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Stage 1: determine initial research question and field of inquiry, stage 2: determine time frame, stage 3: finalize research question(s) to reflect time frame, stage 4: develop search strategy to find relevant articles, stage 5: analyses, stage 6: reflexivity, how to conduct a state-of-the-art literature review.

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Erin S. Barry , Jerusalem Merkebu , Lara Varpio; How to Conduct a State-of-the-Art Literature Review. J Grad Med Educ 1 December 2022; 14 (6): 663–665. doi: https://doi.org/10.4300/JGME-D-22-00704.1

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This article provides a brief introduction to critical steps needed for conducting a high-quality State-of-the-Art (SotA) literature review , one that will add to our understanding of the phenomenon under study. This introduction complements another article in this issue, which discusses the purposes, underlying foundations, strengths, and weakness of SotA reviews in more detail. 1  

The fundamental purpose of SotA literature reviews is to create a 3-part argument about the state of knowledge for a specific phenomenon: This is where we are now. This is how we got here. This is where we could go next ( Table 1 ). Below is a 6-stage process for conducting a SotA literature review. 2   To support this process, questions for guiding each of the 6 stages are provided in Table 2 .

Example of a Medical Education State-of-the-Art (SotA) Literature Review

Six-Stage Approach to Conducting a State-of-the-Art Review With Guiding Questions

It is important to identify individuals who will be a part of the research team. While a SotA review can be conducted by a single author, most SotA reviews harness the perspectives of an interdisciplinary team to generate rich interpretations of the literature. The team should also include a medical librarian to help with developing the search strategy.

In Stage 1, the research team determines the initial research question that incorporates the phenomenon to be addressed in the SotA literature review. It is important to clearly define the field of knowledge and/or practice that will be targeted.

Stage 2 determines the time frame that will define “state-of-the-art” for the research question defined in Stage 1. In Stage 2, the research team should engage in a broad overview of the literature to develop an understanding of the phenomenon's historical development (ie, seminal articles). This process will shape the research team's focus vis-à-vis the pivotal moments in history when the thinking about the phenomenon changed and the time frame for contemporary thinking (ie, the date marking the beginning of this is where we are now thinking). At the end of Stage 2, the research team should be able to justify why a specific year (ie, turning point in history) is chosen to mark the beginning of state-of-the-art thinking around the phenomenon.

Based on the developments from Stages 1 and 2, the research team will revise and finalize the research question(s) to determine what needs to be included in the search strategy and analyses. The revised research question(s) and justification for the timeline must be reported in the article.

Next, a search strategy is developed, enabling the research team to construct the corpus of literature to be included in the SotA review. This involves determining which database(s) to search and when to set the start date for the review. Since the review needs to describe this is how we got here , it must include literature that predates the this is where we are now time frame determined in Stage 2. Stage 4 is an iterative process of testing and revising the search strategy to capture pertinent literature required to meet the purpose of the SotA review. It is important to note that the search goal is not to review all pertinent literature in the SotA review; instead, the goal is to include relevant literature to describe a historical evolution in the field's thinking about a topic. The final search strategy must be included in the manuscript. If possible, a librarian should be consulted when developing the search strategy. A software program such as Covidence may be useful to help organize and share all articles with the research team.

Analysis of the included literature is an inductive process where the research team reads and reflects on the articles and constructs an interpretation of the historical development of how the specific phenomenon is understood in the field. The research team should begin by reading each included article to become familiar with this literature and be able to identify similarities among the articles, ways of thinking that have shaped current understandings, assumptions underpinning changes in understandings over time, and gaps and assumptions in the current knowledge.

Next, the research team can generate the premises that fit the purpose of a SotA review (ie, creating an understanding of the topic, constructing a history of knowledge development that gave rise to this modern thinking, and developing suggestions for future research). In this stage, the research team should highlight specific articles that either support or contradict its premises.

The final step in Stage 5 is to verify the thoroughness and strength of the research team's interpretations. This can be done by selecting different articles and examining if they are congruent with the team's interpretations. The research team may also seek out additional literature that offers alternative interpretations to convey that their summary successfully refutes conflicting interpretations. The goal of this verification work is not to engage in a triangulation process for objectivity or for external confirmation; instead, this process is to help the research team ensure that they have successfully explained their interpretations in a way that supports or refutes the interpretations offered by others.

The SotA manuscript should offer insights into the subjectivity of the research team by describing members who comprise the team, applications of their expertise, and how these informed their interpretations of the data. This reflexivity description will help readers understand the perspectives that informed the interpretation offered by the research team.

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Disclaimer: The opinions and assertions contained herein are those of the authors and are not to be construed as reflecting the views of the Uniformed Services University of the Health Sciences or the US Department of Defense.

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Background/state of the art, background/state of the art.

The background section of the project description must account for the scientific and theoretical framework of the project. Rather than providing an exhaustive description of your entire field of research, you should focus on presenting the information needed to understand your project and situate it in a relevant context.

For this reason, you should also clarify what remains to be understood/done in your field. In other words, you should point out ‘holes’ in existing knowledge – and demonstrate that the aim of your project is precisely to fill these holes.

This section must:

• outline the current state of knowledge within your research field (‘state-of-the-art’) with an emphasis on how your project can fill a hole in the existing research
• demonstrate the scientific rationale for carrying out the project
• if possible, present arguments in support of the project being carried out at this particular time
• if possible, draw attention to your previous contributions to the research field
• maintain a clear and coherent connection to the project’s objectives and perspectives.

How to write a good state of the art: should it be the first step of your thesis?

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A guide to Understanding “State-of-the-Art” Basic Research Techniques in Anesthesiology

Detlef obal.

1 Stanford University School of Medicine, University of Pennsylvania

Andrew McKinstry-Wu

2 Perelman School of Medicine, University of Pennsylvania

Vivianne L. Tawfik

Author contributions: Detlef Obal: This author reviewed the literature, designed figures, wrote chapter on inducible pluripotent stem cell technology, and designed the concept of the review. Andrew McKinstry-Wu: This author reviewed literature, designed figures, and wrote chapter on CRISPR technology. Shaogen Wu: This author reviewed literature, designed figures, and wrote chapter on NGS: next generation sequencing. Vivianne L. Tawfik: This author reviewed literature, designed figures, and wrote chapter on NGS: next generation sequencing. This author helped with the conception of the manuscript. All authors have reviewed the manuscript prior to submission.

Perioperative medicine is changing from a “protocol”-based approach to a progressively personalized care model. New molecular techniques and comprehensive perioperative medical records allow for detection of patient-specific phenotypes that may better explain, or even predict, a patient’s response to perioperative stress and anesthetic care. Basic science technology has significantly evolved in recent years with the advent of powerful approaches that have translational relevance. It is incumbent on us as a primarily clinical specialty to have an in-depth understanding of rapidly evolving underlying basic science techniques in order to incorporate such approaches into our own research, critically interpret the literature and improve future anesthesia patient care.

This review focuses on three important and most likely practice-changing basic science techniques: next generation sequencing (NGS), clustered regularly interspaced short palindromic repeat (CRISPR) modulations, and inducible pluripotent stem cells (iPSC). Each technique will be described, potential advantages and limitations discussed, open questions and challenges addressed, and future developments outlined. We hope to provide insight for practicing physicians when confronted with basic science manuscripts and encourage investigators to apply “state-of-the-art” technology to their future experiments.

NGS: Next-Generation Sequencing for Understanding Cell-Specific Contributions to Your Disease of Interest

In 1977, Frederick Sanger and colleagues developed a sequencing technique based on the incorporation of terminal di-deoxynucleotides that expedited the sequencing of short DNA segments 1 . “Sanger sequencing” has been widely used to identify gene mutations as direct causes or susceptibility factors associated with the development of genetic disorders; however, sequencing occurs one DNA fragment at a time, limiting scalability. During the past decade, next-generation sequencing (NGS) approaches using sample multiplexing (multiple samples in one sequencing run) have been developed that allow for high-throughput screens for genomic mutations and quantification of gene expression in tissue or cells from a disease of interest 2 . Unlike previous assays, NGS techniques directly read sequences of a pool of gene fragments allowing for the detection of novel transcripts, providing unbiased and comprehensive genomic coverage 3 . As a result, NGS approaches have truly revolutionized discovery in a huge variety of science fields (see Figure 1 ).

1) Preparation of Samples: Multiple different types of samples can be processed to obtain material for NGS. RNA is extracted from bulk, laser-capture microdissected (LCM) or fluorescence-activated cell sorted (FACS) tissues. 2) Library construction: RNA is reverse transcribed to cDNA for stability and then fragmented and tagged with specific sequences that allow for batch processing of samples. 3) NGS: Samples are loaded onto a specialized flow cell where amplification and sequencing will take place. Cluster generation forms millions of “DNA colonies” and sequencing-by-synthesis begins to record the sequence of base pairs in each fragment. 4) Bioinformatic analysis: Once sequences are generated, they are aligned to a reference genome, normalized and process for quality control. A host of different analyses can then be applied depending on the outcome of interest. Readers are encouraged to access publicly available NGS datasets and bioinformatics resources for further information. RPKM: reads per kilobase of transcript per million mapped reads; FPKM: fragments per kilobase of transcript per million mapped reads; CPM: counts per million reads mapped; TPM: transcripts per million reads mapped.

Description of the Technique

RNA sequencing (RNA-seq) is the dominant NGS approach used in basic and clinical research. In this review, we will focus on RNA-seq to illustrate the workflow of an NGS-based approach and discuss its advantages and limitations. The overall workflow consists of 4 parts: 1) Preparation of samples, 2) Library construction, 3) Next-generation sequencing, and 4) Bioinformatic analysis.

1) Preparation of samples

The starting materials for RNA-seq study are diverse, including fresh clinical samples, laser capture microdissection (LCM) excised sections from formalin-fixed paraffin-embedded (FFPE) tissue, or cells obtained using fluorescence activated cell sorting (FACS). A standard protocol for tissue collection and RNA purification is critical for successful transcriptomic profiling, because the degradation of RNA can introduce biological bias 4 . Collecting fresh tissue or cells in a commercially available RNA stabilization solution is a practical way to prevent RNA degradation. Newer “single cell” approaches dissociate tissue into single cell suspensions that allow the transcriptome of individual cells to be determined. However, these protocols can also cause cellular stress and transcriptomic changes 5 , 6 , a fact that must be taken into account when designing and interpreting such studies.

2) Library construction

This step converts the sample into a sequencing library that can be sequenced (read) on an NGS instrument. The majority of RNA-seq first requires the preparation of a complementary DNA (cDNA) library for stability. The selection of a specific protocol for subsequent library construction depends on the purpose of the study, for example a ribosomal RNA depletion protocol allows detection of long non-coding RNAs that lack poly-A tails 7 . In general, library preparation involves random fragmentation of the cDNA sample into shorter segments that can be reliably sequenced, followed by ligation of specialized 3’ and 5’ adapters that barcode samples and bind to the NGS flow cell (see below) 8 .

3) Next-generation sequencing

Out of the many types of NGS techniques in the market 9 , the most popular one is termed “sequencing by synthesis” 10 . Generally speaking, each library, consisting of cDNA fragments labeled with adapters, is loaded onto a specialized flow cell with several lanes where amplification and sequencing will take place. Along each lane, there are millions of complementary oligonucleotides that anchor the libraries to the flow cell. Once the fragments have attached, a phase called cluster generation begins to form millions of “DNA colonies". Next, primers and DNA polymerase start adding one fluorescently tagged dNTP base 11 and in each round of synthesis the sequencer records the base added to each fragment on the flow cell in parallel.

There are three major configurations of NGS which should be carefully chosen depending on the study purpose: read length, strand-specificity, and sequencing depth. Read length refers to how many nucleotides can be read from a given library fragment. Longer reads can increase the specificity but depend on sequencer type and may cost more 9 . Paired-end sequencing allows for both ends of the DNA fragment to be sequenced and can increase precision at splicing junctions 12 , repetitive areas of the genome or other difficult-to-sequence segments. Strand specificity refers to sequencing that retains the orientation of the transcripts from 5’ to 3’ end. This technique further increases the accuracy of the results by ensuring that reads are mapped to the correct gene and not a similar gene going in the opposite direction 13 . Sequencing depth refers to how many reads (fragments) are sequenced per sample. A total read depth of 10 to 30 million reads per sample is considered “deep” enough for the coverage of larger transcriptomes such as human or mouse to ensure that low abundant transcripts are detected 9 .

4) Bioinformatic analysis

The output of the NGS process will be millions of sequences that are generated, processed and assigned to each sample, resulting in sequence data on the order of gigabytes. The first step of RNA-seq data analysis should be assessment of the raw sequences. FastQC is a quality control (QC) tool which provides an overview of raw RNA-Seq data [Babraham Bioinformatics, https://www.bioinformatics.babraham.ac.uk ]. To further improve the RNA-seq data quality, tools such as Trimmomatic can be used for trimming and removal of library adapters that were placed for binding to the flow cell 14 . After quality control, sequenced reads are mapped i.e. aligned, to a reference genome or to a transcriptome database. The mapped reads are then counted and normalized for differential expression. Fragments/reads per kilobase of transcript per million mapped reads (“FPKM/RPKM”) or counts per million reads mapped (“CPM”) are some of the units employed to calculate the abundance of each gene expressed in a sample using different algorithms 15 . Transcripts per million (“TPM”) is now becoming popular, which makes it easier to compare the proportion of reads that mapped to a gene in each sample as it first normalizes for gene size followed by read depth 16 , 17 .

The most powerful use of RNA-seq is in finding differentially expressed genes (DEGs) between two or more conditions. There are many tools that perform differential expression calculations including DESeq, edgeR, and Limma-Voom, all of which are available through R/Bioconductor 18 . Most tools use regression or non-parametric statistics to calculate a false discovery rate (FDR) for multiple hypotheses and identify DEGs by log fold change and statistical significance. To obtain a higher-level biological understanding of a list of DEGs, genes can be annotated using Gene Ontology (GO) 19 or Kyoto Encyclopedia of Genes and Genomes (KEGG) terms 20 , which categorize genes based on the family they belong to (e.g. “inflammatory response” or “immune system process”). Downstream Gene Set Enrichment Analyses (GSEA) can then determine whether there are certain pathways or terms (e.g. “circadian rhythms” or “platelet activation”) that are over-represented in experimental vs. control samples 21 . Notably, there are many useful web-based NGS analysis tools available for analysis and visualization of large genomic data (see Figure 3 ). However, we recommend consultation with an experienced bioinformatician to ensure appropriate QC is performed, to reduce the risk of bias and to increase the reproducibility of the results.

Human cardiac and brain tissue is difficult to access and usually not available for drug testing. After reprogramming mature, somatic cells via induction of four transcription factors (Yamanaka factors, O ct4, S ox2/4, K lf4, and M yc), into inducible pluripotent stem cells (iPSC) can be differentiated by modifying cell culture conditions and adding cell-type specific differentiation factors or small molecules into cardiovascular tissue (EC: endothelial cell; CM: cardiomyocytes; CFB: cardiac fibroblasts) or neuronal tissue (Neuro: neurons; AstrC: astrocytes; OligoD: oligodendroglia cells; PeriC: pia cells; MG: microglia cells). Complexity of development and testing increases with the development of organoids or complex engineered tissues. Each tissue type can be subject to functional, morphological, or genetic studies. (scRNA-seq= single cell RNA sequencing).

Advantages and Limitations of NGS technology

NGS techniques, such as RNA-seq, have some clear advantages over older approaches including higher throughput, increased accuracy, ability to detect novel transcripts, higher sensitivity and specificity, and ability to detect low abundance transcripts 22 , 23 . NGS also seems to be the most cost-efficient tool for genome/transcriptome study nowadays. For example, sequencing the entire human genome by NGS now costs no more than $1,000, compared to $100 million in 2001. However, NGS has clear limitations that must be taken into account when designing and interpreting experiments. First, transcription and translation occur at variable rates as does degradation of mRNA and protein. As a result, RNA levels may not necessarily reflect the actual level or activity of proteins of interest. This highlights the need for validation of DEGs at the protein level after RNA-seq profiling. Second, methods of sample preparation may trigger certain cellular pathways (e.g. stress responses) which may be falsely over-represented in the final dataset 24 . Third, batch effects may skew results and experiments need to be carefully designed to mitigate them. Finally, NGS requires a large platform and supercomputer, which might not be easily accessible to all researchers. Importantly, several NGS sequencing services have recently entered the market at a reasonable cost and can be a good choice if internal core facility access is limited.

Implications for Anesthesiologists

In clinical anesthesiology, NGS can be used for the identification of biomarkers for intervention, treatment outcome prediction and understanding disease susceptibility, among other applications 25 , 26 . For example, Tsalik et al. sequenced peripheral blood RNA of 129 representative subjects with systemic inflammatory response syndrome (SIRS) or sepsis 25 . They found that the expression of 338 genes differed between subjects with SIRS and those with sepsis, and the expression of 1,238 genes differed with sepsis outcome (survival vs. non-survival) 25 . They also discovered that the expression of a gene called VPS9D1, which may control cell signaling through endocytosis of intracellular receptors, increased in sepsis survivors, who also expressed a higher number of missense variants in this gene 25 . Another RNA-seq study relevant to our specialty examined ischemic changes, induced by cold blood cardioplegia on the left ventricular myocardium in 45 patients undergoing aortic valve replacement 26 . Through transcriptomic analysis, 1,241 differentially expressed genes were identified when comparing baseline samples to those obtained 79 minutes after aortic cross-clamping 26 . Further functional study of these candidate genes may provide greater insight into the pathophysiology of ventricular ischemia that will guide the development of cardioprotective strategies 26 .

Future Directions of NGS

Many different NGS variants, such as single cell RNA-seq 27 , 28 , spatial transcriptomics 29 , 30 , and 16S rRNA sequencing for microbiome study 31 have been developed. These novel NGS techniques allow researchers to look at the transcriptome heterogeneity and the spatial arrangement of cell types in a given tissue. Using NGS, we are able to uncover new genes of interest, but also to cluster DEGs within key pathways that may be targeted for improved therapeutic efficacy. Furthermore, as additional biomarkers are identified, there is power to monitor disease progression and treatment outcomes. NGS also provides a cost-efficient tool for personalized medicine approaches by identifying whole transcriptome signatures that may allow for mechanism-based treatments. With the development of standardized lab protocols and bioinformatics pipelines, NGS is now becoming more accessible for use in both basic and clinical research. Finally, several publicly available, user-friendly NGS data repositories exist that can be interrogated for hypothesis development or used to cross-reference new datasets 32 . Overall, NGS technologies have already contributed new knowledge to our field and with further adoption by anesthesiologist-scientists, there lies the huge potential for furthering our impact on a diverse set of disease-specific questions.

CRISPR: A Versatile Toolbox from Gene Editing to Expression

Precise genome editing, incorporating animal models, has become an indispensable tool for basic research programs and is increasingly finding use in translational research. The CRISPR (clustered regularly interspaced short palindromic repeats) toolset is a highly adaptable, easily implemented approach for changing a genome at a precise, specified location, i.e. directed recombination. Anesthesia research has incorporated gene editing using various techniques for decades 33 - 36 , and as CRISPR has become the predominant method of editing, it has become central to many anesthesia research endeavors. Uses for this versatile toolset have already extended to include gene regulation, single base editing, and inducible recombination. The future will undoubtedly see a continued expansion of CRIPSR technology with innovations such as conditional or inducible single nucleotide editing on the horizon.

While genetic recombination in mammalian models was pioneered nearly thirty-five years ago 37 , the feasibility (and adoption) of this approach has dramatically increased over the past decade. Early genetic editing relied on rare spontaneous recombination events to incorporate introduced genetic material after untargeted double-stranded DNA (dsDNA) breaks at random locations. Eukaryotes use two separate processes to repair such breaks, homology-directed DNA repair (HDR) or non-homologous end joining (NHEJ.) NHEJ is the faster and more common repair mechanism and involves directly ligating two broken ends of DNA together without the use of any DNA template. This type of repair, as it does not have a template to error check, can result in insertions or deletions at the break site. HDR, in contrast, does use a DNA template to facilitate the and error check the repair process ( Figure 2A .) 38 . By introducing template DNA with a desired change (rather than the native genome, which would normally serve as a template) HDR can be used to insert and change genomic DNA.

A. Short guide RNA (sgRNA) associated with Cas9 pairs with a complementary genomic sequence that is 5’ to a protospacer adjacent motif (PAM.) Because of the genomic target sequence’s proximity to the PAM, the Cas9 cleaves the DNA, creating a double-stranded break (DSB). Repair of the DSB occurs through one of two endogenous processes, non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ can result in short insertions or deletions (indels) of sequence at the break site. Homology-directed repair uses template DNA; if exogenous template DNA is provided that is homologous to the target sequence with a desired sequence inserted at the break site, this directed insertion can be incorporated into the genomic DNA. B. Potential next generation CRISPR technology that has yet to be implemented includes inducible (top) and conditional (bottom) single-base editing without DSB. The example given is a method of inducible base-pair editing using a tetracycline-dependent promoter governing Cas9-nickase fused with a deaminase and guide RNA. The bottom example of conditional single-base editing is the same nCas9-deaminase and sgRNA in reverse orientation with flanking LoxP sites, requiring the presence of Cre recombinase to flip the orientation of the sequence and allow for transcription and translation.

Relying on spontaneous recombination on non-targeted breaks is extremely inefficient with a high chance of gene incorporation at off-target sites 39 , 40 . The ability to specifically target double-stranded DNA (dsDNA) cleavage increases both the reliability and specificity of genome editing. This is despite most double-stranded breaks being repaired using NHEJ rather than the desired template-driven HDR 38 Adaptably targeted DNA breaks expanded the adoption of genome editing due to their higher rate of recombination at desired loci. Targeted breaks were first produced using mega-nucleases, but poor flexibility in target sequence selection of these large proteins caused them to be replaced by chimeric proteins fusing DNA recognition domains with the DNA cleavage domain of the endonuclease Fok I 41 , 42 . This technique requires engineering new chimeric proteins for each new sequence to be targeted. The technical hurdles involved in engineering and cloning novel proteins limited the adoption of techniques employing this strategy

Advantages and Limitations of CRISPR

CRISPR’s major advantages over its gene-editing predecessors are its simplicity and flexibility, while it largely matches the efficacy and efficiency of earlier techniques. The crucial elements of the CRISPR toolbox are derived from an endogenous bacterial antiviral process. In this bacterial system, two short CRISPR RNA sequences (crRNA), one of which includes a 17-28 nucleotide portion homologous to bacteriophage sequences, form complexes with CRISPR-associated (Cas) proteins to target the viral sequence encoded by the crRNA 43 . When such a complex incorporates a DNA-cleaving Cas element, such as Cas9, the result is a targeted double-stranded DNA break (DSB) with the target specified by an RNA sequence. The viral genomic sequence to be targeted must include a short protospacer adjacent motifs (PAMs), a 3-7 nucleotide long sequences specific to Cas enzyme, 3’ to the target sequence for the CRISPR-Cas complex to attach cleave the DNA. Though these systems are native to bacteria, with modifications to Cas9 to include a nuclear localization sequence, they are able to modify mammalian genomic DNA as well 44 - 46 . Another early modification to the system was the substitution of a single guide RNA (sgRNA) for the two crRNAs, further reducing complexity 47 . The modular nature of the system, with interchangeable Cas protein elements (for enzymatic activity) and sgRNAs (for target sequence selection) combined with the comparative ease of nucleotide synthesis make this system an extremely useful biotechnology tool, both flexible in its application, and simple enough to implement that it can be readily adopted without extensive technical prerequisites.

CRISPR/Cas9 was the first widely adopted CRISPR toolset for gene editing. Despite its simplicity and flexibility, this approach is not without its drawbacks. While RNA-mediated targeting for CRISPR/Cas9 is flexible and easily changed, genomic locations for cleavage are limited by the necessity of a PAM sequence 3’ to the cleavage site. PAM sequences are short and occur frequently enough within the genome that this restriction rarely presents a major impediment, but it is a limitation. Like their predecessors, zinc-finger nucleases and TALENs, CRISPR/Cas9 systems make use of endogenous DNA repair processes after DSBs. The two separate events, the initial DSB and subsequent repair, are each possible sources of error. Though targeting is specified with PAMs and the 17-20 nucleotide sequence in crRNA, careful examinations of off-target effects shows that CRISPR systems display some flexibility in their targeting of the sequence, causing DSBs at sites not predicted by sequence alone 48 , 49 . These off-target cleavages can produce insertions and deletions at disparate sites within the genome. Even when a DSB occurs in the correct location, NHEJ, with its associated insertions of random genetic material or deletions of proximate sequence, occurs eight times more frequently than the more desirable HDR 50 . Numerous improvements have made to CRISPR/Cas genome-editing systems, largely focused on increased targeting flexibility, decreased off-target cutting, and increasing the rate of HDR over NHEJ after a cut. Increased targeting flexibility through altering PAM sequences is a significant source of innovation. The most commonly employed Cas9, derived from Streptococcus pyogens, has NGG as its PAM sequence, but Cas derived from other species have alternate sequences, expanding potential targets within a genome. In addition to naturally-occurring homologs, engineered Cas variants can have altered, and even more flexible, PAM requirements 51 - 53 . Diverse strategies have been successful at decreasing the rate of off-target DSBs, another goal for innovation. Cas variants specifically engineered to decrease the rate of off-target cutting 54 , altering sgRNA length, or delivering the complete sgRNA-Cas ribonucleoprotein complex (rather than a plasmid encoding both) have all succeeded at reducing off-target cutting 55 , 56 . Using Cas nickases, which cut only one of the two DNA strands, and two sgRNAs independently targeting sequences directly opposite each other, two cuts need to independently occur to create a DSB, significantly reducing the rate of off-target DSBs 57 . Finally, temporal and spatial control of enzymatic activity using light- or small-molecule-activated Cas can also decrease off-target cutting 58 , 59 . Similarly, diverse approaches have been deployed toward increasing the rate of template driven HDR and decreasing the incidence of NHEJ after producing a DSB. The rate of HDR can be increased using either small molecule adjuvants or single-stranded DNA templates 60 , 61 . Certain Cas variants produce staggered DSBs in DNA, leaving overhanging sticky ends. These staggered breaks have a higher rate of HDR, such that use of this class of Cas, including Cpf1, can increase the rate of successful recombination 62 .

Applications of CRISPR-based systems have expanded beyond simple facilitated recombination using targeted DSBs. Fusing catalytically inactive (dCas9) to other functional domains has emerged as a flexible means of targeting specific loci in the genome for a variety of purposes. These include attaching gene regulatory elements to dCas9 to enhance or inhibit transcription 63 , 64 , fluorescent tags to visualize gene locations in the nuclei of live cells 65 , and methyltransferase to alter local DNA methylation 66 . Newer techniques have opened the door to gene editing without creating DSBs. The fusion of Cas nickase with various deaminases allows the direct conversion of C·G > T·A or A·T > G·C and removal of the opposite paired base, such that by targeting the correct strand, any single genomic base pair can be converted to any other 67 , 68 . This technology is still in its early stages, however, and deaminases can induce significant off-target mutations 69 . The potential for such “second generation” CRISPR-based gene editing nevertheless has yet to be fully realized. Specifically, approaches that have been previously used in conjunction with first generation CRISPR gene editing technology, inducible and conditional expression, could be applied to second generation systems to produce cell-line-specific or inducible targeted single-base mutations ( Figure 2B ) Such limited targeted mutations could allow for a range of studies on critical genes that otherwise prove lethal when altered during development.

The use of CRISPR technology for gene editing has only become widely adopted over the past five years, so we are just beginning to see studies using CRISPR-engineered mutants in the anesthesiology 70 . As CRISPR-based gene-editing and advanced CRISPR techniques become the foundation for a wide variety of investigations in the science and practice of anesthesiology, it will become ever more critical for anesthesiologists to understand many of the previously mentioned hazards and potential limitations of the technique, in addition to appreciating the diverse applications. In interpreting these types of studies, it is important that readers look for appropriate controls accounting for potential off-target cutting including insertions and deletions. Alternatively, the study authors may include of measures to minimize the impact of such events such as backcrossing animals to a wild-type background, or the use of enzymes or adjuvants that minimize such off-target events.

Future Directions of CRISPR

CRISPR-based technologies have significantly lowered the barrier to entry for gene editing approaches, and new applications offer similar potential for gene regulation and epigenomic approaches. CRISPR has already broadened the types of basic science questions that can be asked by opening the door to the use of new model organisms 71 . Separately, second generation editing has the potential to bridge gene editing technologies into the clinical realm in the coming years through precise and limited genomic alterations. We have yet to see the full scope of the impact of these powerful and flexible approaches, but continued refinement with reduction of off target effects will be necessary before this technology can move from the bench to the bedside.

iPSC: Induced Pluripotent Stem Cell Technology – a Road to Personalized Medicine

Human-embryonic stem cells (hESC) are derived from the inner cell mass of fresh or frozen embryos at the blastocyst stage of development 72 . Embryonic stem cells (ESCs) are self-renewable and able to give rise to cells of all three germ layers (ectoderm, endoderm, and mesoderm) 73 , 74 . Indefinite replication makes hESCs a valuable tool to study anesthetic mechanisms in human tissue. However, ethical controversies and limited supply of donor human embryos restricted the use of this cell type and alternative approaches were warranted.

In 2006, Takahashi and Yamanaka performed an experiment, in which they selected 24 different transcription factors as candidates to induce pluripotency in somatic cells, i.e. reprogramming of already differentiated cells into ESC-like cells 75 . After extensive screening, they identified four factors, i.e. Oct2/4, Sox2, Klf4, and c-Myc (also known as “OSKM”) which were essential to produce ESC-like colonies able to form teratoma 75 . These induced pluripotent stem cells (iPSCs) have become a valuable tool in basic science and only 6 years after his original observation, Shinya Yamanaka (together with John B. Gurdon) received the Nobel Prize in Physiology or Medicine for their “discovery of reprogramming mature cells into pluripotent stem cells”.

The great potential of this technique relies on its ability to generate any somatic cell line out of an indefinitely dividing stem cell pool, specific to a particular patient and collectable without invasive procedures. In other words : Peripheral human blood cells can be reprogrammed into stem cells which will be subsequently differentiated into a somatic cell of any type 76 - 81 . With regard to morphology, surface marker expression, global gene expression profile, DNA methylation status, and other characteristics, hiPSC and hESC are considered to be similar 82 . Herein we will focus on hiPSC-derived somatic cells, which are of particular interest for anesthesiologists: cardiomyocytes and neurons ( Figure 3 ). Although, hiPSC-derived somatic cells are frequently cultured in mono-layers (2D culture) when used for drug- and phenotype testing, our description will concentrate on hiPSC-derived cardiac (hiPSC-CM) and cerebral (hiPSC-NC) tissues grown as organoids (i.e. 3D structures), which are build out of different cell types to better reflect the “natural” original.

To build these 3D structures (organoids) based on an patients’ own cells has been a long-term objective within the iPSC community; however, the organoids should reflect structure and cell-composition of their natural counterparts. Nowadays, structure modifications have been archived by applying different bioengineering methods 83 , 84 and by modifying cell culture conditions 85 , 86 . Diversity of cell-composition within the organoids has been realized in two different ways: Human iPSC/ESC have been differentiated by stepwise adding small molecules and growth factors resulting in subsequent development of various cell types reflecting “naturally” formed organoids 87 . On the other side, hiPSC have been differentiating into multiple cell types separately and then in a subsequent step “assembled” to the final organoid. While the first method allows to study “natural” progression of cell differentiation as during organogenesis, the second method allows to control for cell-type composition within the organoids superior for description of cell-cell interactions.

Compared to 2D cell layers, hiPSC growing as 3D organoids have less contact with culture plates, thus favoring interaction between cells and maintaining histological and genetic complexity even after long-term culture 88 . Importantly, cells grown in organoids have a distinct gene expression profile compared to their 2D counterparts resulting in contrasting pharmacological profiles: Luca and colleagues demonstrated that tumor cells cultured in a 3D model express significantly less epithelial growth factor receptors (EGFR, a factor important for controlling cell proliferation) compared to tumor cells cultured in 2D culture which made these cells less susceptible to EGFR tyrosine kinase inhibitors 89 As EGFR tyrosine kinase inhibitors are tested as chemotherapeutic agents, the therapeutic efficiency reported in different studies may deviate significantly and impact drug development.

HiPSC-Derived Cardiac Organoids

By aligning hiPSC-CM in parallel rather than randomly within an extracellular matrix 90 , Zimmermann, Eschenhagen and colleagues developed engineered heart tissue (EHT) models in which cardiomyocytes increased in size, and started to form a connected syncytium. EHT allows advanced functional assessment of human tissue in a dish 91 , even in a high-throughput screening format 92 . The current end of these bioengineering developments constitutes a 3D electro-mechanically coupled, fluid-ejecting miniature ventricle derived from hiPSC-CM 93 , in which anesthetic agents and other drugs might be tested under “physiological” conditions “in vitro” in the future.

HiPSC-Derived Cerebral Organoids

HiPSC-derived cerebral organoids offer great potential to study the neurotoxic effects of anesthetics and their mode of action. Currently, the time to generate self-organized 3D structures spans from 50 to 100 days 94 , 95 . The construction of neuronal organoids 96 , 97 allows the prediction of anesthetic-induced neurotoxicity 98 and with advancements in single-cell technology, we have the tools to obtain transcriptomes of single cells and to cluster cells with identical transcriptional activities 99 . With the combination of hiPSC-NC and vascular cells, neuronal-vascular units have been developed 100 , 101 to study brain development 102 or function and neurotoxicity of anesthesia-inducing drugs 103 , 104 . Unfortunately, this technique is still under-utilized in our specialty and during anesthetic and analgesic drug development.

Advantages and Limitations of iPSC technology

Although hiPSC have been discovered more than 10 years ago, the technique is still evolving and some limitations should be considered: Retrieving cells from patients appears to be relative easy and straightforward, however, as the efficiency of reprogramming primary cells (skin fibroblast vs. blood cells) depends on the cell type 105 , which should therefore be carefully chosen.

Another consideration for using hiPSC technology in clinical practice may be the maturation status of hiPSC derived cardiomyocytes or neurons. Although functional cardiomyocytes (i.e. contracting cells, hiPSC-CM) develop within 7-9 days 106 , an additional of 90-120 days may be required to generate cardiomyocytes with a more mature phenotype (i.e. expression of adult genes, normal morphology and expression of normal T-tubule and sarcomere structure) 107 . Similarly, differentiation hiPSC towards neuronal tissue requires between two 108 - 110 and six weeks 111 - 113 . These culture requirements may currently not be suitable for “pre-operative” bed-side tests but may impact care for patients undergoing long-term planned elective, high-risk procedures or for patients with repeated anesthetic exposures (i.e. pediatric patients after correction of congenital heart defects).

Additional concerns about hiPSCs’ genetic stability during reprogramming, as well as differentiation-dependent variability and heterogeneity exist 114 , 115 . These reservations may be justified; however, they reflect limitations of a relatively young technology which will be resolved within the coming years.

How do hiPSC-derived cells help anesthesiologists to understand cellular function and where are the limitations? By using a few assays as an example, advantages and limitations of iPSC-based techniques will be outlined:

One of the advantages of hiPSC-CM and hiPSC-NC is the ability to perform functional assessments of patient specific tissue in vitro . Cardiac arrhythmias and QTc interval prolongation are significant milestones to overcome during drug development. With the development of hiPSC-CM, drug-induced QTc prolongation can be successfully studied in human tissue in a dish . hiPSC-CM of patients with inherited channelopathies can be easily generated from a 10 ml blood sample to study ion channel function or membrane potential changes by different methods (i.e. patch clamp, fluorescence dyes, genetically introducing voltage, and/or calcium indicators 116 - 119 ).

Most recently, hiPSC-CM have been used to determine the cardiac side profile of KSEB01-02, a new compound with hypnotic properties 120 . Using a high-throughput screening method, KSEB01-02 turned out to be less cardio-depressant than propofol, elucidating the advantage of using hiPSC-CM to screen for cardiovascular side effects of a new drug in human heart tissue.

Similar methods can by utilized to evaluate communication within human neuronal networks: HiPSC-NC become electrical active within a few days of differentiation 121 , and with new methods like “patch-seq” it is possible to study simultaneously functional (patch-clamp) and genetic (i.e. single-cell RNA sequencing) characteristics of single cells of a human neuronal network in vitro 122 , 123 .

Another advantage of hiPSC-CM and hiPSC-NC is the ability to measure cell toxicity of anesthetic agents: Propofol infusion syndrome is a life-threatening complication of long-term sedation with rather high doses of propofol 124 . Nevertheless, the mechanism of cell toxicity was unclear on the cellular level. By treating hiPSC-CM with propofol (0-50 ug/ ml) for 48 hours, Kido and colleagues revealed that high propofol concentrations cause mitochondrial dysfunction by downregulating of PGC-1-α in human tissue 125 . While the majority of neurotoxicity studies are based on non-human tissue, hiPSC-NC also offered an opportunity to study the impact of prolonged isoflurane exposure on neuronal survival and neurogenesis in human tissue 126 . Interestingly, the anesthetic toxicity seem to be strongly dependent on cytosolic Ca 2+ concentration within the hiPSC-NC 126 .

The molecular mechanism of some anesthetic actions are still unknown: Ketamine’s recent renaissance as antidepressant agent raised questions about the underlying mechanism of action: Collo and colleagues differentiated hiPSCs into floor plate-derived midbrain dopaminergic neurons and demonstrated that the structural plasticity and expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPAR) and their subunit GluR1 and GluR2 might be causative for positive, antidepressant effects of the drug 127 .

While anesthetic mechanisms have been studied in non-human tissue samples or in large clinical trials, these examples show that with the new hiPSC technology a unique venue became available to study functional and toxicological effects of modern and future anesthetics in human tissues. By using patient specific iPSC-derived tissues, personalized medications as well as therapeutic plans can be developed and transferred into clinical practice.

Future Directions of iPSCs

While iPSC technology has facilitated the development of new agents in cardiovascular medicine 128 , 129 , neurology 130 , and oncology 131 , the use for development of new perioperative medications has been limited. Although anesthesia related drugs haven’t changed dramatically over the past 50 years, the fascinating developments in molecular and cell-based techniques will pave the way towards improved anesthesia drugs focusing precisely on the individual patient. Not only will iPSC technologies allow the development of new compounds, but also facilitate our understanding of current anesthesia medications. With the rapid progress in biochip technology, we will hopefully advance to generating testing platforms which will shorten the time to determine patient-specific personalized drug cocktails during the perioperative care period.

Basic research techniques are rapidly evolving and increasing in complexity and sophistication. Although anesthesia is considered safe, we should not ignore the opportunity to utilize such approaches to study diseases of relevance to our specialty, to increase our understanding on how anesthetic drugs function and where possible to move forward drug development opportunities and personalized medicine. This review will hopefully encourage others to describe their sophisticated research tools to a) make them more understandable and relevant to the clinically practicing anesthesiologist and b) to increase the interest and facilitate the use of these exciting novel techniques to improve anesthesia care in the future.

Summary Statement:

This review will summarize cutting-edge basic science research techniques, including inducible stem cell (iPSC) technologies, gene editing with CRISPR/Cas9, and finally next generation sequencing. Included chapters will describe advantages, limitations, and importance of each technique for anesthesiologists.

Acknowledgments

D.O. was supported by a Seed Grant of the Stanford CV Institute and the Transdisciplinary Initiatives Program (TIP) of the Stanford Maternal & Child Health Research Institute, institutional and/or departmental resources, as well as grant funds from the National Institutes of Health (NIH) Grant K08NS094547 (V.L.T.), K08GM123317 (A.M.-W.) and the Rita Allen Foundation Award in Pain (V.L.T.).

Glossary of Terms

Prior Presentations: Portions of the content of this review have been presented in seminar format during the 2019 IARS annual meeting in Montreal, Canada.

Disclosures

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Writing the Research Proposal: The Art and the Science

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A research proposal is a document containing details about the research which is to be undertaken. It should be self-contained and start with a fundamental enquiry related to the research questions(s) and the hypothesis (es) on which it is based. The objectives and key questions are the fundamental pillars of a research proposal and formal grant application.

Research is to see what everybody else has seen, and to think what nobody else has thought — Albert Szent-Györgyi, Hungarian Biochemist (1893–1986) No Research without Action, No Action without Research —Kurt Lewin, German-American psychologist (1890–1947)

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Writing a Research Proposal

Research Going Forward?

Basic Research

1 what is a research proposal.

A study protocol, often used for clinical trials, is a document that describes the objectives, elucidates the methodology, ethical considerations (consent process) as well as the overall execution template to guide the research [ 1 ]. Journals dedicated to studying protocols also encourage publication of research protocols prior to the wrap-up and conclusion of studies to encourage transparency and avoid duplication of research.

A research proposal can be in a free form or follow a suggested format, usually prescribed by the science funding body or organization. In general, a proposal format includes:

Background and Rationale

Aims and Objectives of the study

Details of the Research Question

Methodology

Ethical considerations

Details of the primary investigator, co-investigator. Internal and external collaborators.

Estimated Budget

1.1 The Title

The title should be composed of key substantive words, which may include the characteristics and geographical location of research, the sample population as well as a hint of the result. The title may at times be interrogative.

For example, the title of this study protocol— Study protocol of a cluster randomized controlled trial to evaluate the effectiveness of a system for maintaining high-quality early essential newborn care in Lao PDR [ 2 ]. conveys the study design, i.e., the cluster randomized trial, the purpose of the research and also the study population and site of the research.

Many grant organizations have specific requirements for space and characters for titles, so ensure conformity with those.

1.2 Abstract

Some organizations require an abstract or an executive summary at the beginning of the research proposal. This is a brief precis and description of your research project, background, methods, and analytical plan.

1.3 Background and Rationale

What is already known about this research area? Have there been any previous studies already addressing this issue? Many research funding bodies expect the investigators to cite a systematic review related to the subject area, typically the most credible review available on the subject. The literature review ought to highlight why the research question being addressed in the project is important.

1.4 Aims and Objectives of the Research

The primary goals of the research study are described in terms of its aims and objectives. Aims are general and broad statements that state the intent of the researchers and what they hope to achieve, while objectives are more specific that describe the path to achieving those aims.

Aims and Objectives are encouraged to follow the SMART criteria:

Specific— Precision about what is going to be done

Measurable— Outcomes clearly defined

Attainable— Can be possibly achieved and not overly ambitious

Realistic— Possible in the presence of available resources, e.g., finances, time, and manpower

Time Constrained— Bound by time

1.5 Details of the Research Question

The PICOT format ( P opulation, I ntervention, C ontrol or comparison group, O utcome of disease, T ime, or T ype of study) helps to frame a good research question.

1.6 Methodology

The Methodology section follows the background and literature review so should organically follow through with a recapitulation of your research question, the related scholarly research available, and your own aims and objectives [ 3 ].

This section attempts to answer the 5 W and One H questions—Who, What, When, Where, Why and How.

This section should be written carefully in full detail in such a way that anyone who reads it can replicate the experiment; if it is a new statistical model that you propose, you should be able to apply it to your own dataset. This section is like a road map that helps the investigator to navigate the planned study and should be written in the future tense. The methodology section should not just state the methods chosen, but should appropriately justify why it was selected based on sound scholarly research. It should also discuss the limitations in the proposed methods and compromises made based on existing constraints, for example, the choice of sampling and the inclusion and exclusion criteria. This section should include the following details [ 3 ]:

1.6.1 Study Area/Location

This subsection states the institutional and departmental affiliation or the site where the research is to be conducted. If it is clinical research, data on the patient enrolment area, patient catchment area and patient recruitment area, and whether they were from the outpatient or inpatient departments.

In other instances, with population-based research, the study population may be a community, region, or even a larger population aggregate, comprising the universe of the study from which subjects will be recruited.

1.6.2 Study Population

The enrolment should always be according to a pre-defined population. The inclusion and exclusion criteria to be used for the study should also be mentioned in this section.

For example, ‘to study the prevalence of lymphoma in Sjogren’s syndrome’, the inclusion will include all known cases of Sjogren’s syndrome and those with a histologically confirmed diagnosis of lymphoma. The exclusion criteria will be lymphoma associated with HIV, hepatitis B and C viral infections, or due to other causes.

1.6.3 Sample Size

The number of cases to be recruited into the study should be stated here. The formula that is intended to be used to derive the sample size should also be included as well as the statistical power, the expected prevalence of the disease, or the shift in outcome if relevant. Recent statistical software has made this step much easier so the statistical software employed for these calculations, e.g., EPI INFO should be mentioned.

1.6.4 Study Design

The designs are broadly experimental or observational and may utilize primary or secondary data. Differences between both are tabulated in Table 12.1 [ 4 ].

1.6.5 Study Duration

The date of initiation and expected completion date are mentioned. The study can start only after the institutional board gives ethical clearance.

1.6.6 Methodology of the Trial

In this section, the step-by-step approach of the study is described. It could begin with patient recruitment and the patient consent approval process and could be followed by a description of the physical examination and a systemic examination to be carried out. It should include whether there are tests to be done in the intervention and the length of period after which any change in the intervention will be studied.

1.6.7 Outcome of the Disease

This is the most important consequence of the study. Mention what change you are expecting during the experiment. For any disease, the outcome can be:

Complete recovery

Incomplete recovery

There may also be disease-specific outcome measures like in rheumatology one can use a visual analogue scale, ESR, CRP, Disease Activity Score (DAS), Simplified Disease Activity Index, and Clinical Disease Activity Index.

1.6.8 Data Collection

The data collected may be qualitative or quantitative. A protocol for data collection which incorporates and satisfies the requirements of Good Clinical Practice (GCP) should be included. For example, in a study around the effect of pomegranate juice on lipid profile and type 2 diabetes, the systolic and diastolic blood pressures, and the lipid profile of the patients were measured at baseline and end-line following 12–14 h of fasting (5).

Furthermore, a data analysis plan, a data quality assurance plan, a statistical plan, and a contingency plan for missing and spurious data should also be discussed.

2 What Are Study Designs?

Experimental studies are further divided into randomized controlled trials or nonrandomized trials. The observational studies are of four types, cohort studies, case–control studies, cross-sectional, and ecological studies (Fig. 12.1 ).

Overview of types of study design

3 What Is an Intervention?

The intervention can be in the form of treatment using a drug, vaccine, or even a dietary supplement, the use of a diagnostic or therapeutic procedure or the introduction of an educational tool (Table 12.2 ).

4 What Are the Various Type of Trials with Interventions?

There are various types of intervention-based trials that are listed below:

Randomized controlled trials

In this, a patient in one arm of the trial gets the intervention and the others get the placebo or the standard of care. Randomized Controlled Trials are considered the gold standard in clinical research, and evidence generated is considered to be the highest in the hierarchy of evidence.

Nonrandomized trials

In this, the intervention is given only to some participants, e.g., those who can afford the drug or vaccine and the others get standard care.

Interventional studies without concurrent controls

If a new drug is to be given in a particular condition to a study population, historical controls are used (Fig. 12.2 ).

Study with external controls

Pre- and post-intervention study

An example is the use of an educational tool applied in a specific population - the change is noted before and after the intervention.

Factorial studies

In this, two or more interventions are examined and their effects are studied collectively and separately.

Crossover studies

Each patient is given one type of intervention and then after a predefined time a washout period is instituted where controls and experimental subjects are swapped and then given the other trial intervention (Fig. 12.3 ).

RCT crossover design

Cluster randomization trials

The intervention in a single person may be easy to apply but once the trial is done on a community it requires to be studied in a cluster of the population. Units of randomization are clusters, and not individuals. By design, they are large and complex studies but more definitive in terms of health population and systems research.

In the cluster randomized trial in Matiari and Hala in Sindh Pakistan (Fig. 12.4 ), clusters were allocated to intervention and control groups through stratified randomization. The intervention package included health promotive MNCH activities through community healthcare workers and this RCT. Neonatal mortality rate was lower in intervention clusters at 43.0 deaths per 1000 live births and 49.1 per 1000 in control groups (RR 0.85, 0.76−0.96; p  = 0.02) [ 5 ].

Cluster randomized trial in Matiary and Hala in Sindh Pakistan

4.1 Observational Studies

4.1.1 cross-sectional study.

These studies represent data collected over a defined period of time, and are often used to calculate the prevalence of a certain disease or condition (Fig. 12.5 ). However, a major limitation is that they do not help establish causality due to unknown temporality. They are also vulnerable to recall error and response bias when questions about the past are assessed.

Cross-sectional study (Basic design)

4.1.2 Cohort Studies

This study design is longitudinal in nature and involves following a group of participants who have a particular characteristic in common, for example, they may all be in the same geographical region or in a particular occupation (Fig. 12.6 ). The study involves following them prospectively and studying associations between exposure and outcome.

Cohort study (Basic design)

4.1.3 Case–Control Studies

By definition, case studies are observational and retrospective. Also known as ‘case-referent studies’. They are observational because they compare patients who have the disease or outcome of interest (cases) with patients who do not have the disease or outcome (controls) and look back retrospectively to compare how frequently the exposure to a risk factor is associated with the disease (Fig. 12.7 ).

Retrospective study

Case–control studies are observational because no intervention is attempted and no attempt is made to alter the course of the disease. The goal is to retrospectively determine the exposure to the risk factor of interest from each of the two groups of individuals: cases and controls. These studies are designed to estimate odds.

4.1.4 Ecological Studies

Ecological studies the association between exposure and disease, but the unit of observation is a community or even a broader regional area such as a country. The ecological study design is relatively cheaper as it often utilizes published population-level statistics such as mortality and morbidity estimates.

5 What Are the Ways to Do Randomization?

5.1 the methods of randomization in a clinical trial.

Blinding refers to keeping trial subjects, doctors, and trial-related persons or data collectors unaware of the allocated intervention to eliminate bias.

Various methods of randomization are used:

Simple randomization—Using techniques like a pack of cards, flipping of a coin (even—control, odd—treatment), or throwing dice (e.g., below and equal to 3—control, over 3—treatment) can be used.

Random allocation is done using random number tables.

Block randomization—This method is designed to randomize subjects into groups.

Stratification randomization—This is a two-stage procedure where the first randomization is done according to clinical features which may influence the outcome and in the second a particular treatment is given to that arm.

Minimization—The aim of minimization is to minimize the imbalance between the number of patients in each treatment over a number of factors.

6 What Are Other Considerations Before Conditional Trial?

6.1 ethical considerations.

IRB approval must be discussed and the data collection protocol’s adherence to the World Medical Association Declaration of Helsinki on Ethical Principles for Medical research involving Human Subjects should be discussed.

6.2 Clinical Trial Registration

Clinical trials are registered mainly to avoid publication bias and selective reporting . In addition, clinical trial registries serve to increase transparency and access to clinical trials for the public. There have been recently much efforts to increase standardization of registration of clinical trials with the WHO aim to ‘achieving consensus on both the minimal and the optimal operating standards for trial registration’. The largest and most frequently used repository of trials is ClinicalTrials.gov , run by the United States National Library of Medicine (NLM). Others include Australian New Zealand Clinical Trials Registry (ANSCTR), the International Standard Randomised Controlled Trial Number in the UK, and the Clinical Trials Registry in India.

6.3 Details of Primary Investigator, Co-Investigator, Internal and External Collaborators

Rich and salubrious scholarship is obtained through active collaborations between multiple centres of research. Individual and institutional collaborations should be listed.

This is an estimation of the expenses which are likely to be incurred during the study period. It should include travel costs, costs of reagents, instrumentation, special software, and manpower. Each cost should be justified and if the research is expected to run for several years, the inflation cost should also be added.

7 What Are the Various Extramural Source of Funding for Doctors for a Research Project?

Usually, there are two mechanisms for grants, i.e., sole-source grants commissioned grants for pre-qualified organizations, and competitive grants. Sole source grants are non-competitive with contractual arrangements whereas competitive grants are managed through call for proposal where individuals or organizations are required to go through a competitive process. The various funding agencies are given below. Besides the government authorities, many speciality associations also offer funding for MD, DM projects. Many universities and colleges have travel grants for students who present papers in national or international conferences. The details and age limits and inclusion criteria need to be checked on their websites (Table 12.3 ).

8 Conclusions

The application for study proposal should be well-structured and free from any grammatical and scientific errors.

Not all research projects receive funding.

For protocol and research grants, the methodology should be written in the future tense.

Use references in this section for the definition of various terms related to your study.

Write comprehensively describing minute details of the experiment.

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Department of Surgical Gastroenterology and Liver Transplantation, Sir Ganga Ram Hospital, New Delhi, India

Samiran Nundy

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Institute for Global Health and Development, The Aga Khan University, South Central Asia, East Africa and United Kingdom, Karachi, Pakistan

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Nundy, S., Kakar, A., Bhutta, Z.A. (2022). Writing the Research Proposal: The Art and the Science. In: How to Practice Academic Medicine and Publish from Developing Countries?. Springer, Singapore. https://doi.org/10.1007/978-981-16-5248-6_12

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Why and how to write the state-of-the-art.

State-of-the-art (SoTA) is a step to demonstrate the novelty of your research results. The importance of being the first to demonstrate research results is a cornerstone of the research business. You cannot get a Nobel prize (anymore) by learning Einstein ‘s law of photoelectric effect by heart and presenting it as your own. Einstein did it before you, and everyone knows it because he published it. When Einstein published his theory the theory had novelty. Einstein could demonstrate his theory’s novelty by presenting a SoTA and showing that no other researcher had ever presented such results. That’s why he got a Nobel prize and you will not.

Besides demonstrating the novelty of your research results, a SoTA has other important properties:

• It teaches you a lot about your research problem. By reading literature related to your research problem you will learn from other researchers and it will be easier for you to understand and analyze your problem.
• It proves that your research problem has relevance. If many people are trying to solve the same research problem as you, and if you can demonstrate this in your SoTA, then no one can tell you the problem you are trying to solve is not important.
• It shows different approaches to a solution. By seeing many different approaches taken by other researchers, you can evaluate your own approach and realize its novelty (or lack of it) easily. You can also see which approaches are the most popular and which are dead ends.
• It shows what you can reuse from what others have done. Especially when doing research on new software, it is amazing how many people have made the exact software you are planning to make. Just do a search on sourceforge and github .

So how to write a good SoTA? Writing a good SoTA is 110% dependent on having a clear problem definition . If you have failed in defining your problem clearly, you will fail in writing a good SoTA. The reason is that you will not know what related research you should investigate. So if you have problems with your SoTA, please go back and work on your problem definition! Here are some steps/hints on starting to write:

• SoTA is not a one-way road. You will not sit down one evening and write your SoTA. You will do it all the time while writing your paper/report. Knowing what other researchers are doing should be a part of your life for all the duration of your research. So an important step is to create a system of registering and summarizing what you read. Use some bibliography software such as Mendeley , BibTeX or EndNode or Zotero , register everything you read, and register your understanding of what you read, in your own words.
• Be critical when choosing your literature. Don’t read everything. There is a LOT of garbage out there on the web, and you don’t want to waste your time on garbage. One important criteria for choosing your literature is to make sure that it is peer-reviewed and is already presented/published in well-known conferences/journals. In case of technical IT-related stuff, ACM and IEEE are good places to start (do searches in Engineering Village ). It is also a good idea to set up an initial SoTA literature list together with your supervisor.
• Stop reading! Make an initial selection of literature (10-20 papers, depending on research problem) and stick to these for a while. Don’t go on finding new papers all the time, or you will never finish your thesis!
• Spend time on analysis and not on making summaries! A mere summary of 10-20 papers is not a SoTA. There is software out there that can summarize any paper for you, automatically and much faster than you ever will be able to. Your summaries become a SoTA only when you relate the SoTA papers to your own problem analysis.
• Always give credit! Not giving credit for others’ research is also called plagiarism .
• For more advanced writers: It is always a good practice to document your methodology for doing state-of-the-art survey. This means you should document how you searched for literature, what literature you included and what you excluded, how you did your analysis and so forth. This is called systematic review, and a de facto guide for doing systematic reviews in the field of software engineering is available here . You can also find many useful links on wikipedia .

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Writing proposals for research funding is a peculiar facet of North American academic culture, and as with all things cultural, its attributes rise only partly into public consciousness. A proposal’s overt function is to persuade a committee of scholars that the project shines with the three kinds of merit all disciplines value, namely, conceptual innovation, methodological rigor, and rich, substantive content. But to make these points stick, a proposal writer needs a feel for the unspoken customs, norms, and needs that govern the selection process itself. These are not really as arcane or ritualistic as one might suspect. For the most part, these customs arise from the committee’s efforts to deal in good faith with its own problems: incomprehension among disciplines, work overload, and the problem of equitably judging proposals that reflect unlike social and academic circumstances.

MAIN SECTIONS

• Capture the reviewer’s attention
• Aim for clarity
• Establish the context
• What’s the pay-off?
• Use a fresh approach
• Describe your methodology
• Specify your objectives

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Research Proposal Writing for Fine Arts and Music

• Where to Start?

Phase 1- Go deep!

Phase 2- give yourself some structure, phase 3- writing your research question, phase 4- finalising your research question, research methodologies, key research texts.

• Graduate Research
• Talk to a Librarian

Where to Start- Forming a Research Question

Your creative project is well underway and now it is time to research and write about it. But how do you turn your ideas into a  research question?

Use the tabs on this page to work through  Phases 1, 2, 3 and 4 . These may help you identify themes and questions you would like to explore in your project, and how to turn these into successful library search strategies.

The library also has many resources available to help you learn more about Practice-Based Research and other research methodologies. There are links to these in the  Research Methodologies  and  Key Research Texts  tabs.

1) Reflect on your current creative project. Write down your motivations and aims for creating the project. Write down any significant discoveries you have made along the way. 

2) Ask yourself, have there been any issues arising from the project that I could research? These issues may be based around the concept, themes or technical aspects of your work or a combination of these. 

3) If you are struggling to identify any issues, talk with peers who know your work and ask them for feedback. Explain to them your motivations, aims and discoveries. Ask them if your work effectively achieves what you set out to do. Write down any issues related to your work that come from this discussion. 

4) Based on the issues you and your peers have identified, select one or two issues that are the most interesting and important for you. These issues will form the basis of your research question. 

1) Using the 1 or 2 issues you have identified, you can narrow the focus of your research question. Here are a few questions to ask:

• Time period (Is my work related to a specific period?)
• Person (Is my work related to a specific director, composer, choreographer, performer or writer?)
• Technical tool (Does my work use any particular technique?)
• Social or political issue (Does my work address a social issue such as war, disease, love, race or sexuality?)
• Contemporary issue (Does my work explore an issue in contemporary music theatre? Eg. music, writing, performance venues, lyrics, acting or dancing)
• What kind of role does my creative project have in the broader context of contemporary music theatre?

2) Make a list of responses to these questions. Select which responses are most interesting and relevant to your creative project.

You will now have a narrower idea for your research question.

1) Play around with your research question. Write it down as a question or statement in a number of different ways. Try to get to at least ten different statements, but no pressure! Not all of them will be good. You might:

• Change around the phrasing of the issue
• Change your original words for synonyms
• Say the question out loud
• Explain it to one of your peers and write down your explanation.

2) Highlight the questions that seem clearest to you. 

3) Forget about your question or topic for 24 hours. Instead, reflect on your creative project, watch some documentation or perform part of the project.

4) Return to your list of questions with fresh eyes. Make a list of the best three questions/topic sentences. If you have already identified that one question is the best one for you, stick with that one. 

1) For each question, spend 10 minutes searching  Discovery . Use an  Information Search Planner  to help you search effectively (download one from the link below). 

2) Assess the results of your searches as you go and use these results to help you choose one of your three questions. Ask yourself:

• Is there a lot of information available on this topic?
• Has my question already been answered?
• Who is writing about the topic of my question?
• Which of my searches is finding results that are the most interesting and relevant to my creative project?

3) By answering the questions above, you will be able to select a suitable question.  If not, reassess your question and repeat Phase 2 onwards. Alternatively, you may wish to discuss your question with your lecturer or supervisor.

REMEMBER:  Your question will change over time. When you are making work and researching, your ideas will change and your question can too!

Research Methods

The Library collections offer many resources on research methods. Doing a simple keyword search in the  Library Catalogue  will give you a list of many. You can use the keywords below as a starting point:

• Practice-led research
• Practice based research
• Research-led practice
• Artistic research

You can also  modify your search  to limit it to resources held at Southbank Library, or to our Online Resources if you're interested in an ebook.

Subject Headings in the Catalogue

The links below will launch a search in the library catalogue for specific subject headings:

Arts -- Research

Qualitative Research

Quantitative Research

Mixed Methods Research

Research Method

SAGE Research Methods

SAGE Research Methods is a research tool to help you design your research project.  Search by keywords or use the visual interface in the  Methods Map . Its main focus is research in the social sciences.

• SAGE Research Methods SAGE Research Methods is a research methods tool which links SAGE’s renowned book, journal and reference content with truly advanced search and discovery tools. Researchers can explore methods concepts to help them design research projects, understand particular methods or identify a new method, conduct their research, and write up their findings. SAGE Research Methods Cases is a collection of case studies of real social research that faculty can use in their teaching. Cases are original, specially commissioned, and designed to help students understand often abstract methodological concepts by introducing them to case studies of real research projects.

• Next: Graduate Research >>
• Last Updated: Apr 9, 2024 4:34 PM
• URL: https://unimelb.libguides.com/proposalwritingcreativearts

An example of state-of-the-art using Open Innovation

How to achieve a state of the art?

How to realize a state-of-the-art? How can Open Innovation help? Some simple answers below.

State-of-the-art : definition

A state of the art is the identification of previous knowledge to avoid reinventing. Making a state-of-the-art makes it possible to verify or justify that one produces new knowledge, for a thesis of doctorate or the filing of a patent, for example. The state-of-the-art often also includes the identification of actors – academic or industrial – who are at the origin of knowledge : the “ecosystem”. Better, this ecosystem can be questioned to complete and strengthen the state-of-the-art. This is where Open Innovation comes in. Especially since the algorithms of data mining and classification of Open Innovation platforms allow an acceleration of the research tasks for publications and actors which are often tedious. Let’s have a deeper look to a state-of-the-art example.

State-of-the-art example: dead leaves

For this state-of-the-art example, let’s take a real case treated by an industrial company we know: the problem of fallen leaves on train tracks in autumn . The dead leaves cause a loss of adhesion between the rails and the wheels of the trains, in particular because of the transformation of the leaves at the passage of the trains. The transformed material causes a loss of adhesion between the wheel and the rail which requires lengthening braking distances, and hence disrupts the timing of the trains.

Start with scientific publications

The state-of-the-art will consist of multiple queries using a search engine. We recommend starting with an investigation of scientific publications (which are often richer and more explicit than patent sources). In our example, this exploration begins with a combination of keywords such as “wheel”, “rail”, “leaves”,”adhesion”, which will return publications on these topics.The interesting publications are saved.

• >>Read also: Better, Faster, Harder – SME Open Innovation

Identify keywords & draw a mindmap

Find interesting patents.

In a second step, the most interesting queries are exploited with patent data sources. In addition to identifying interesting patents in the field, they help better understand the ecosystem. In our case, a very rich university ecosystem (and, to a lesser extent, industrial) has appeared in Europe (Great Britain, Germany, Ukraine, Netherlands, Italy, …), in Asia (China, South Korea, Japan) and in North America (United States, Canada).

Contacting experts: a specific approach to Open Innovation

Finally, to complete the state-of-the-art, the industrial company decided to contact a number of university groups. This step, which is optional, is also specific to an Open Innovation approach. It has the advantage of giving the possibility to talk to specialists in the field who can update the state-of-the-art with the latest and even unpublished research data. Even through a conversation lasting less than an hour, it is possible to identify the important points or to project oneself in the future, which a purely bibliographic search allows only in a very limited way . It’s like going to an annual conference and interviewing the world’s most famous experts !

Contacting experts: asking the right questions

A final remark. In any case, before contacting an expert to refine a state-of-the-art, one must ask himself “why would he/she spend time on my subject ?”. In our case on dead leaves, a large corporation was at the origin of the question and many experts are interested in a dialogue that can lead to collaboration. But other options are possible : a simple exchange of information, the possibility of setting up a joint project, remuneration, etc.

A fair / balanced relationship is one of the keys to succeed in Open Innovation projects.

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Research Challenge Grant winners announced

• Author By Deborah Fox
• April 26, 2024

Eight research projects involving 16 faculty members are the winners of the inaugural Dean’s Research Challenge Grants. Proposals submitted this year were required to focus on one of two themes: “Equity” or “Environment.”

Established in 2023 to encourage innovative scholarly work, the College of Arts and Sciences committed $47,000 to this initiative.

“Supporting faculty research is a top priority,” said Dr. Heather Dillaway, dean of the College of Arts and Sciences. “In the College of Arts and Sciences, we view the University’s motto of ‘Gladly we learn and teach,’ as being directly intertwined with research excellence. The purpose of universities, in the purest sense, is to create knowledge and share knowledge. That means we must invest in both research and teaching.”

The 2023-2024 winning proposals are:

Awards: Equity

• Michael Hendricks (POL) along with co-PIs Noha Shawki (POL), Joan Brehm (SOA) and Eric Peterson (GEO)- Cleaning up the river without clearing out the neighborhood: Floating gardens in the Chicago River
• Dan Ispas (PSY) along with co-PI Alexandra Ilie (PSY)- Exploring equity in personnel selection: Investigating differential prediction and applicant reactions to personality tests
• Maura Toro-Morn (SOA-LALS) along with co-PI Jim Pancrazio (LAN) – project aims to assess and capture the experiences of COBAS program participants
• Susan Chen (ECO) –  gender and race disparities in the impact of COVID-19 on job market outcomes

Awards: Environment

• Ben Wodika (BIO) along with co-PIs Vickie Borowicz , Matt Dugas , and Sydney Metternich – Sugar Creek Urban Ecology Area Committee Application: Do species matter? A test of the Functional Equivalence Hypothesis using Ground Beetles
• Christopher Mulligan (CHE) – Next Generation analytical methods for on-site and on-demand environmental pollutant monitoring
• Sudipa Topdar (HIS)- Lifeless Feathers, Masculinity, and Ecological Imperialism in the Late Colonial Himalayas (1800-1947)
• Maochao Xu (MAT)-Cyberbullying Dynamics: A Predictive modeling exploration in college communities

Award recipients will deliver presentations about their research projects at a College of Arts and Sciences Research Symposium on Friday, October 11. Additional details about this event will be forthcoming.

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