Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity.

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. Here, to better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 44 published data sets. We find strong evidence that gRNA sequence dependent activity is largely influenced by the ability of the Cas9/gRNA complex to find the target site rather than activity at the target site and that this drives sequence dependent differences in gRNA activity between different species. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.

Applications and future directions for optical coherence tomography in dermatology.

Optical coherence tomography (OCT) is a noninvasive optical imaging method that can generate high-resolution en face and cross-sectional images of the skin in vivo to a maximum depth of 2 mm. While OCT holds considerable potential for noninvasive diagnosis and disease monitoring, it is poorly understood by many dermatologists. Here we aim to equip the practising dermatologist with an understanding of the principles of skin OCT and the potential clinical indications. We begin with an introduction to the technology and discuss the different modalities of OCT including angiographic (dynamic) OCT, which can image cutaneous blood vessels at high resolution. Next we review clinical applications. OCT has been most extensively investigated in the diagnosis of keratinocyte carcinomas, particularly basal cell carcinoma. To date, OCT has not proven sufficiently accurate for the robust diagnosis of malignant melanoma; however, the evaluation of abnormal vasculature with angiographic OCT is an area of active investigation. OCT, and in particular angiographic OCT, also shows promise in monitoring the response to therapy of inflammatory dermatoses, such as psoriasis and connective tissues disease. We additionally discuss a potential role for artificial intelligence in improving the accuracy of interpretation of OCT imaging data.

What is AI? Applications of artificial intelligence to dermatology.

In the past, the skills required to make an accurate dermatological diagnosis have required exposure to thousands of patients over many years. However, in recent years, artificial intelligence (AI) has made enormous advances, particularly in the area of image classification. This has led computer scientists to apply these techniques to develop algorithms that are able to recognize skin lesions, particularly melanoma. Since 2017, there have been numerous studies assessing the accuracy of algorithms, with some reporting that the accuracy matches or surpasses that of a dermatologist. While the principles underlying these methods are relatively straightforward, it can be challenging for the practising dermatologist to make sense of a plethora of unfamiliar terms in this domain. Here we explain the concepts of AI, machine learning, neural networks and deep learning, and explore the principles of how these tasks are accomplished. We critically evaluate the studies that have assessed the efficacy of these methods and discuss limitations and potential ethical issues. The burden of skin cancer is growing within the Western world, with major implications for both population skin health and the provision of dermatology services. AI has the potential to assist in the diagnosis of skin lesions and may have particular value at the interface between primary and secondary care. The emerging technology represents an exciting opportunity for dermatologists, who are the individuals best informed to explore the utility of this powerful novel diagnostic tool, and facilitate its safe and ethical implementation within healthcare systems.

Spatial constraints govern competition of mutant clones in human epidermis.

Deep sequencing can detect somatic DNA mutations in tissues permitting inference of clonal relationships. This has been applied to human epidermis, where sun exposure leads to the accumulation of mutations and an increased risk of skin cancer. However, previous studies have yielded conflicting conclusions about the relative importance of positive selection and neutral drift in clonal evolution. Here, we sequenced larger areas of skin than previously, focusing on cancer-prone skin spanning five decades of life. The mutant clones identified were too large to be accounted for solely by neutral drift. Rather, using mathematical modelling and computational lattice-based simulations, we show that observed clone size distributions can be explained by a combination of neutral drift and stochastic nucleation of mutations at the boundary of expanding mutant clones that have a competitive advantage. These findings demonstrate that spatial context and cell competition cooperate to determine the fate of a mutant stem cell.

A dynamic landscape of allergen associations in delayed-type cutaneous hypersensitivity.

BACKGROUND: Delayed-type hypersensitivity represents a significant clinical and public health challenge. Patients undergoing patch testing may exhibit positive reactions to more than one allergen. It is recognized that reactions to specific pairs of allergens are associated, reflecting a combination of exposure patterns and structural similarity. OBJECTIVES: To explore the influence of time of testing, age, sex and atopy status on allergen pair associations in a series of 45 110 consecutive patients tested over 30 years. METHODS: Patch test records of all patients undergoing testing with a modified European baseline series between 1985 and 2014 were retrieved from a database at St John's Institute of Dermatology. Reactions were read on days 2 and 4. For each allergen it was recorded whether the allergen was tested and whether the result was positive or negative. RESULTS: This is the largest reported study of patch test allergen pair relationships. Our analysis shows a high degree of variability in allergen pair associations. Rigorous statistical analysis reveals a large number of differences between groups, including a significant increase in the association between formaldehyde and multiple formaldehyde-releasing preservatives over the study period, in addition to pair associations with cobalt and formaldehyde-releasing preservatives. These were present to a significantly greater extent in men than in women. CONCLUSIONS: These observations extend our understanding of cutaneous allergy, with implications for both clinical practice and mechanisms of cutaneous hypersensitivity.

Applications of platelet-rich plasma in dermatology: A critical appraisal of the literature.

Platelet-rich plasma (PRP) is an autologous blood-derived product enriched in platelets, growth factors, chemokines and cytokines. Initial applications were predominantly in musculoskeletal and maxillofacial fields, however in recent years, it has been used for a range of dermatological indications including wound healing, fat grafting, alopecia, scar revision and dermal volume augmentation. Here, we critically appraise the literature relating to the usage of PRP within Dermatology. We have evaluated in vitro data, preclinical animal studies and human trials. We conclude that, whilst the literature may be consistent with a modest benefit for specific indications, there is not sufficient evidence supporting the efficacy of PRP to justify a role in routine dermatological practice at the present time. However, since PRP is generally well tolerated with few reported complications, further study may be justified in the context of organized trials.

Disseminated coxsackievirus A6 affecting children with atopic dermatitis.

Coxsackievirus A6 (CV-A6) is an emerging pathogen that has in recent years been associated with atypical hand, foot and mouth disease. This manifests as a generalized papular or vesicular eruption, which may be associated with fever and systemic disturbance. We report a series of six children presenting to a single centre in the UK with disseminated CV-A6 infection on a background of atopic dermatitis (AD). Our patients exhibited a widespread papular or vesicular eruption in association with exacerbation of AD. Several of our cases mimicked eczema herpeticum, but the extent was more generalized, and individual lesions were discrete rather than clustered and were less circumscribed in character. This series highlights that CV-A6 infection may be encountered in the UK, and should be considered in the differential diagnosis of an acute exacerbation of AD, particularly in children.

Identification of a 21 amino acid peptide conferring 3-hydroxypropionic acid stress-tolerance to Escherichia coli.

We report the identification of a novel small open reading frame in Escherichia coli. The sORF (called iroK) encodes a 21 amino cid peptide, which when translated confers a 133% (ca. 20 g/L) increase in resistance to 3-hydroxypropionic acid. We show that iroK conferred tolerance is additive to previously identified tolerance mechanisms involving relief of inhibited metabolism, yet does not involve altered 3-HP transport. This result demonstrates the continued surprises that microbial genomes hold and emphasize the importance of comprehensive discovery methods in future strain and metabolic engineering efforts.

Rapid dissection of a complex phenotype through genomic-scale mapping of fitness altering genes.

The understanding and engineering of complex phenotypes is a critical issue in biotechnology. Conventional approaches for engineering such phenotypes are often resource intensive, marginally effective, and unable to generate the level of biological understanding desired. Here, we report a new approach for rapidly dissecting a complex phenotype that is based upon the combination of genome-scale growth phenotype data, precisely targeted growth selections, and informatic strategies for abstracting and summarizing data onto coherent biological processes. We measured at high resolution (125 NT) and for the entire genome the effect of increased gene copy number on overall biological fitness corresponding to the expression of a complex phenotype (tolerance to 3-hydroxypropionic acid (3-HP) in Escherichia coli). Genetic level fitness data were then mapped according to various definitions of gene-gene interaction in order to generate network-level fitness data. When metabolic pathways were used to define interactions, we observed that genes within the chorismate and threonine super-pathways were disproportionately enriched throughout selections for 3-HP tolerance. Biochemical and genetic studies demonstrated that alleviation of inhibition of either of these super-pathways was sufficient to mitigate 3-HP toxicity. These data enabled the design of combinatorial modifications that almost completely offset 3-HP toxicity in minimal medium resulting in a 20 g/L and 25-fold increase in tolerance and specific growth, respectively.

Parallel mapping of genotypes to phenotypes contributing to overall biological fitness.

Laboratory selection is a powerful approach for engineering new traits in metabolic engineering applications. This approach is limited because determining the genetic basis of improved strains can be difficult using conventional methods. We have recently reported a new method that enables the measurement of fitness for all clones contained within comprehensive genomic libraries, thus enabling the genome-scale mapping of fitness altering genes. Here, we demonstrate a strategy for relating these measurements to the individual phenotypes selected for in a particular environment. We first provide a mathematical framework for decomposing fitness into selectable phenotypes. We then employed this framework to predict that single-batch selections would enrich primarily for library clones with increased growth rate, serial-batch would enrich for a broad collection of clones enhanced via a combination of increased growth rate and/or reduced lag times, and that overlap among selected clones would be minimal. We used the SCalar Analysis of Library Enrichments (SCALEs) method to test these predictions. We mapped all genomic regions for which increased copy number conferred a selective advantage to Escherichia coli when cultured via single- or serial-batch in the presence of 1-naphthol. We identified a surprisingly large collection (163 total) of tolerance regions, including all previously identified solvent tolerance genes in E. coli. We show that the majority of the identified regions were unique to the different selection strategies examined and that such differences were indeed due to differences among enriched clones in growth rate and lag times over the solvent concentrations examined. The combination of a framework for decomposing overall fitness into selectable phenotypes along with a genome-scale method for mapping genes to such phenotypes lays the groundwork for improving the rational design of laboratory selections.

A genomics approach to improve the analysis and design of strain selections.

Strain engineering has been traditionally centered on the use of mutation, selection, and screening to develop improved strains. Although mutational and screening methods are well-characterized, selection remains poorly understood. We hypothesized that we could use a genome-wide method for assessing laboratory selections to design selections with enhanced sensitivity (true positives) and specificity (true negatives) towards a single desired phenotype. To test this hypothesis, we first applied multi-SCale Analysis of Library Enrichments (SCALEs) to identify genes conferring increased fitness in continuous flow selections with increasing levels of 3-hydroxypropionic acid (3-HP). We found that this selection not only enriched for 3-HP tolerance phenotypes but also for wall adherence phenotypes (41% false positives). Using this genome-wide data, we designed a serial-batch selection with a decreasing 3-HP gradient. Further examination by ROC analysis confirmed that the serial-batch approach resulted in significantly increased sensitivity (46%) and specificity (10%) for our desired phenotype (3-HP tolerance).

Selective pressure for a decreased rate of asymmetrical divisions within stem cell niches may contribute to age-related alterations in stem cell function.

Most mammalian continuously renewing tissues are maintained by stem cells located within stem cell niches. Each niche contains a number of stem cells that replicate asymmetrically to give differentiated cells and also undergo periodic deletion and compensatory replacement by symmetrical "self-renewal" divisions of stem cells remaining within the niche. It has been recognized that there is selective pressure for an increased rate of self-renewal divisions and that the increasingly likely consequence is neoplasia. However, to date it has not been appreciated that there is also an independent selective pressure for a decreased rate of asymmetrical divisions. In this article, the origin of this second type of selective pressure is explained and its consequences explored through the use of computer modeling. It is shown that age-related changes in a range of mammalian stem cell compartments can be understood in the context of a decreased rate of asymmetrical stem cell divisions with an increased propensity for self-renewal divisions. It is proposed that a decreased rate of asymmetrical divisions impairs the ability of old stem cell compartments to respond effectively to stress.

The role of cellular senescence may be to prevent proliferation of neighboring cells within stem cell niches.

It has long been suspected that cellular senescence is an anticancer mechanism; however, it has been difficult to understand the advantage for the organism of retaining mutant cells in a postmitotic state rather than simply deleting them by apoptosis. It is proposed that in certain circumstances apoptosis promotes neoplasia by causing cells adjacent to the deleted cell to divide and that the role of cellular senescence is to prevent this. This may be particularly important in mammalian stem cell niches. After loss of a stem cell from a niche, another stem cell within the same niche divides symmetrically to restore the original number. The most important human malignancies arise from tissues maintained by stem cells, and there is increasing evidence that stem cells are the targets for at least the initial genetic changes that occur during carcinogenesis. If a subset of stem cells within a niche arises containing an oncogenic mutation, then tumor suppressor mechanisms promote apoptosis of these cells, and the niche restores the original number of stem cells by replication of both normal and mutated stem cells. Thus, paradoxically apoptosis increases turnover of mutant cells with associated risk of further genetic changes. However, if in addition mutant cells can become senescent, then the niche is progressively filled by senescent cells until either the mutant cells are eliminated or the niche is completely occupied by postmitotic cells, thereby preventing further evolution of the neoplastic clone. The consequences of this hypothesis are explored by computer modeling.

Malignant eccrine poroma in breast cancer-related lymphoedema.

We describe the first case of malignant eccrine poroma arising in a lymphoedematous site. The patient had long-standing lymphoedema of the upper limb following breast cancer treatment. Lymphoedema is a recognised complication of breast cancer treatment and a risk factor for the subsequent development of malignancy. Possible mechanisms for this are discussed.

Tutor and student perceptions of the tutor's role in problem-based learning.

The role of the tutor in problem-based learning (PBL) differs dramatically from the traditional teaching role with which dental students and faculty are familiar. In this investigation, the dimensionality and complexity of the tutor role were assessed by first- and second-year dental students and by the tutors themselves using a twenty-four-item Likert-type questionnaire that served as one component of a comprehensive web-based assessment tool, the PBL-Evaluator. Evaluations were completed after each PBL case over a period of one and a half years by one class of students and for half a year by a second class. Exploratory principal components analyses of the responses to the questionnaire revealed a more complex factor structure for tutors than for students. While a five-factor solution was required for the tutors, a simpler two-factor solution sufficed for both groups of first-year students, and a three-factor solution was necessary for the second-year students. Although tutors displayed a more nuanced perspective on their performance than did students, items related to tutor modeling of professional conduct within tutorials consistently emerged as important for both.

The expanded Trail Making Test: rationale, development, and psychometric properties.

Many procedures have been employed to determine the specific cognitive components necessary for successful Trail Making Test (TMT) performance. Yet, there is still considerable disagreement in the literature as to what these components might be. The present study explores an alternative methodology to address this problem. By systematically varying the stimuli within the TMT format, it may eventually be possible to isolate the cognitive demands of this test. As the first step toward this goal, two experimental forms of the TMT, forms X and Y, were developed and subjected to empirical validation. The results indicate that this Expanded Trail Making Test possesses adequate concurrent and criterion validity to support the proposed methodology. The results also suggest that the psychometric properties of the TMT format are robust to alterations in test stimuli. Secondary benefits of this methodology, in terms of explaining between-group variance and in terms of cross-cultural assessment, are discussed.