Determining Effective Drug Concentrations for Selection and Counterselection Genetics In Drosophila melanogaster

We recently integrated into fly genetics a set of four selection and two counterselection markers and their corresponding drugs that can be used individually or in combination. These markers eliminate the need to visually screen progeny. Before using these markers in new genetic backgrounds, effective selection/counterselection concentrations should be established for each marker/drug combination. This protocol describes how to set up, perform, and analyze a drug titration curve to determine the effective selection/counterselection drug concentrations for their corresponding markers. For complete details on the use and execution of this protocol, please refer to Matinyan et al., 2021

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] Bac Clones for Selection Transgenesis and Functional Gene Analysis Using Drosophila melanogaste

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker whit

Synthetic Assembly DNA Cloning to Build Plasmids for Multiplexed Transgenic Selection, Counterselection or Any Other Genetic Strategies Using Drosophila melanogaster

We recently described a drug-based selectable and counterselectable genetic platform for the animal model system Drosophila melanogaster, consisting of four resistance and two sensitivity markers that allow direct selection for, or counterselection against, a desired genotype. This platform eliminates the need to identify modified progeny by traditional laborious screening using dominant eye and body color markers, white+ and yellow+, respectively. The four resistance markers permit selection of animals using G418 sulfate, Puromycin HCl, Blasticidin S, or Hygromycin B, while the two sensitivity markers allow counterselection of animals against Ganciclovir or Acyclovir, and 5-Fluorocytosine. The six markers can be used alone or in combination to perform co-selection, combination selection and counterselection, as well as co-counterselection. To make this novel selection and counterselection genetics platform easily accessible to and rapidly implementable by the scientific community, we used a synthetic assembly DNA cloning platform, GoldenBraid 2.0 (GB2.0). GB2.0 relies on two Type IIs restriction enzymes that are alternatingly used during successive cloning steps to make increasingly complex genetic constructs. Here we describe how to perform synthetic assembly DNA cloning using GB2.0 to build such complex plasmids, using the assembly of both components of the binary LexA/LexA-Op overexpression system, a G418 sulfate-selectable LexA transactivator plasmid, and a Blasticidin S-selectable LexA-Op responder plasmid, as an example. We demonstrate the functionality of these plasmids by including the expression pattern obtained after co-injection, followed by co-selection using G418 sulfate an Blasticidin S, resulting in co-transgenesis of both plasmids. Protocols are provided on how to obtain, adapt, and clone DNA parts for synthetic assembly cloning after de novo DNA synthesis or PCR amplification of desired DNA parts, how to assemble those DNA parts into multipartite transcription units, followed by how to further assemble multiple transcription units into genetic constructs of increasing complexity to perform multiplexed transgenic selection and counterselection, or any other, genetic strategies using Drosophila melanogaster. The protocols we present can be easily adapted to incorporate any of the six selectable and counterselectable, or any other, markers to generate plasmids of unmatched complexity for various genetic applications. A protocol on how to generate transgenic animals using these synthetically assembled plasmids is described in an accompanying Current Protocols article (Venken,Matinyan, Gonzalez, & Dierick, 2023). Basic Protocol 1: Obtaining and cloning a de novo synthesized DNA part for synthetic assembly DNA cloning. Basic Protocol 2: Obtaining and cloning a DNA part amplified by PCR from existing DNA resources for synthetic assembly DNA cloning. Alternate Protocol 2: Obtaining, adapting, and cloning a DNA part amplified by PCR from existing DNA resources for synthetic assembly DNA cloning. Basic Protocol 3: Synthetic assembly DNA cloning of individual DNA parts into a multipartite transcription unit. Basic Protocol 4: Synthetic assembly DNA cloning of multiple transcription units into genetic constructs of increasing complexity

A 3D quantitative method for analyzing bone mineral densities: a case study on skeletal deformities in the gilthead sea bream, <i>Sparus aurata</i> (Linnaeus, 1758)

Skeletal deformities, one of the major threats for aquaculture, have been studied extensively. These include opercular malformations in gilthead sea bream (Sparus aurata), a key fish species for Mediterranean aquaculture. What is causing it and at what morphogenetic level it arises, however, is still unclear. Here we focus on bone formation, at the level of bone mineralization. Several methods have been used to study bone mineralization density (BMD), however, these are frequently limited when targeting a high-resolution, three-dimensional mapping of BMD. We used micro-computed tomography (micro-CT) data to perform such a 3D quantification of BMD levels in gilthead sea bream that showed different levels of opercular bone deformations. This approach has the advantage of not having to rely on calibration phantoms, as long as relative BMD values are needed. The results show an increased BMD in deformed opercles compared to normal ones, especially in a bilaterally-deformed specimen. Furthermore, we show that opercular deformations are not necessarily associated with similar mineralization patterns in other mineralized cranial elements, except for the otoliths. Also, mineralization seems to occur left-right independently, matching earlier observations of such an independency of the opercular phenotype as a whole. This study confirms that a quantitative characterization of BMD patterns in 3D is feasible, even in smaller specimens, and that it has several advantages over other commonly used approaches

Linking experiences of child sexual abuse to adult sexual intimate partner violence: the role of borderline personality features, maladaptive cognitive emotion regulation, and dissociation

Stress and Psychopatholog

A prospective cohort study assessing clinical referral management & workforce allocation within a UK regional medical genetics service

Abstract Ensuring patient access to genomic information in the face of increasing demand requires clinicians to develop innovative ways of working. This paper presents the first empirical prospective observational cohort study of UK multi-disciplinary genetic service delivery. It describes and explores collaborative working practices including the utilisation and role of clinical geneticists and non-medical genetic counsellors. Six hundred and fifty new patients referred to a regional genetics service were tracked through 850 clinical contacts until discharge. Referral decisions regarding allocation of lead health professional assigned to the case were monitored, including the use of initial clinical contact guidelines. Significant differences were found in the cases led by genetic counsellors and those led by clinical geneticists. Around a sixth, 16.8% (109/650) of referrals were dealt with by a letter back to the referrer or re-directed to another service provider and 14.8% (80/541) of the remaining patients chose not to schedule an appointment. Of the remaining 461 patients, genetic counsellors were allocated as lead health professional for 46.2% (213/461). A further 61 patients did not attend. Of those who did, 86% (345/400) were discharged after one or two appointments. Genetic counsellors contributed to 95% (784/825) of total patient contacts. They provided 93.7% (395/432) of initial contacts and 26.8% (106/395) of patients were discharged at that point. The information from this study informed a planned service re-design. More research is needed to assess the effectiveness and efficiency of different models of collaborative multi-disciplinary working within genetics services. Keywords (MeSH terms) Genetic Services, Genetic Counseling, Interdisciplinary Communication, Cohort Studies, Delivery of Healthcare, Referral and Consultation

Relative contribution of mutations in genes for autosomal dominant distal hereditary motor neuropathies: a genotype-phenotype correlation study

Distal hereditary motor neuropathy (HMN) is a clinically and genetically heterogeneous group of disorders affecting spinal α-motor neurons. Since 2001, mutations in six different genes have been identified for autosomal dominant distal HMN; glycyl-tRNA synthetase (GARS), dynactin 1 (DCTN1), small heat shock 27 kDa protein 1 (HSPB1), small heat shock 22 kDa protein 8 (HSPB8), Berardinelli-Seip congenital lipodystrophy (BSCL2) and senataxin (SETX). In addition a mutation in the (VAMP)-associated protein B and C (VAPB) was found in several Brazilian families with complex and atypical forms of autosomal dominantly inherited motor neuron disease. We have investigated the distribution of mutations in these seven genes in a cohort of 112 familial and isolated patients with a diagnosis of distal motor neuropathy and found nine different disease-causing mutations in HSPB8, HSPB1, BSCL2 and SETX in 17 patients of whom 10 have been previously reported. No mutations were found in GARS, DCTN1 and VAPB. The phenotypic features of patients with mutations in HSPB8, HSPB1, BSCL2 and SETX fit within the distal HMN classification, with only one exception; a C-terminal HSPB1-mutation was associated with upper motor neuron signs. Furthermore, we provide evidence for a genetic mosaicism in transmitting an HSPB1 mutation. This study, performed in a large cohort of familial and isolated distal HMN patients, clearly confirms the genetic and phenotypic heterogeneity of distal HMN and provides a basis for the development of algorithms for diagnostic mutation screening in this group of disorder

Delegation of GP-home visits to qualified practice assistants: assessment of economic effects in an ambulatory healthcare centre

<p>Abstract</p> <p>Background</p> <p>Against the background of a decreasing number of general practitioners (GPs) in rural regions in Germany, the AGnES-concept (AGnES = GP-supporting, community-based, e-health-assisted, systemic intervention) supports the delegation of regular GP-home visits to qualified practice assistants. The concept was implemented and evaluated in different model projects in Germany.</p> <p>To explore the economic effects of this concept, the development of the number of home visits in an ambulatory healthcare centre was analysed and compared with the number of home visits in the surrounding county.</p> <p>Methods</p> <p>Information about GP-home visits was derived from reimbursement data of the ambulatory healthcare centre and a statutory health insurance. Information about home visits conducted by AGnES-practice assistants was collected from the project documentation over a time period of 12 consecutive quarter years, four quarter years before the beginning of the project and 8 quarter years while the project was implemented, considering background temporal trends on the population level in the study region.</p> <p>Results</p> <p>Within the ambulatory healthcare centre, the home visits by the GPs significantly decreased, especially the number of medically urgent home visits. However, the overall rate of home visits (conducted by the GPs and the AGnES-practice assistants together) did not change significantly after implementation of the AGnES-concept. In the surrounding county, the home visit rates of the GPs were continuous; the temporal patterns were approximately equal for both usual and urgent home visits.</p> <p>Conclusion</p> <p>The results of the analyses show that the support by AGnES-practice assistants led to a decrease of GP-home visits rather than an induction of additional home visits by the AGnES-practice assistants. The most extended effect is related to the medically urgent home visits rather than to the usual home visits.</p

Fractal analyses reveal independent complexity and predictability of gait

Locomotion is a natural task that has been assessed for decades and used as a proxy to highlight impairments of various origins. So far, most studies adopted classical linear analyses of spatio-temporal gait parameters. Here, we use more advanced, yet not less practical, non-linear techniques to analyse gait time series of healthy subjects. We aimed at finding more sensitive indexes related to spatio-temporal gait parameters than those previously used, with the hope to better identify abnormal locomotion. We analysed large-scale stride interval time series and mean step width in 34 participants while altering walking direction (forward vs. backward walking) and with or without galvanic vestibular stimulation. The Hurst exponent α and the Minkowski fractal dimension D were computed and interpreted as indexes expressing predictability and complexity of stride interval time series, respectively. These holistic indexes can easily be interpreted in the framework of optimal movement complexity. We show that α and D accurately capture stride interval changes in function of the experimental condition. Walking forward exhibited maximal complexity (D) and hence, adaptability. In contrast, walking backward and/or stimulation of the vestibular system decreased D. Furthermore, walking backward increased predictability (α) through a more stereotyped pattern of the stride interval and galvanic vestibular stimulation reduced predictability. The present study demonstrates the complementary power of the Hurst exponent and the fractal dimension to improve walking classification. Our developments may have immediate applications in rehabilitation, diagnosis, and classification procedures