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In a European project about formative assessment, Local Working Groups (LWGs) from six participating countries made use of a format for teacher-researcher collaboration. The activities in each LWG involved discussions and reflections about implementation of four assessment formats. A key aim was close collaboration between teachers and researchers to develop teachers' formative assessment practices, which were partially evidenced with changes in attributes of self-efficacy. The research question was: to what extent do working with formative assessment strategies in collaboration with researchers and other teachers differentially affect individual self-efficacy beliefs of practicing teachers across different educational contexts? A 12-item teacher questionnaire, with items selected from a commonly used international instrument for science teaching self-efficacy, was distributed to the participating teachers before and after their work in the LWGs. A novel method of analysis using networks where participants from different LWGs were linked based on the similarities of their answers, revealed differences between empirically identified groups and larger super groups of participants. These analyses showed, for example, that one group of teachers perceived themselves to have knowledge about using formative assessment but did not have the skills to use it effectively. It is suggested that future research and development projects may use this new methodology to pinpoint groups, which seem to respond differently to interventions and modify guidance or instruction accordingly.

Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae's metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches.

Biological computer-aided design and manufacturing (bioCAD/CAM) tools facilitate the design and build processes of engineering biological systems using iterative design-build-test-learn (DBTL) cycles. In this book chapter, we highlight some of the bioCAD/CAM tools developed and used at the US Department of Energy (DOE) Joint Genome Institute (JGI), Joint BioEnergy Institute (JBEI), and Agile BioFoundry (ABF). We demonstrate the use of these bioCAD/CAM tools on a common workflow for designing and building a multigene pathway in a hierarchical fashion. Each tool presented in this book chapter is specifically tailored to support one or more specific steps in a workflow, can be integrated with the others into design and build workflows, and can be deployed at academic, government, or commercial entities.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

Recent research and reform efforts in science education have consistently stressed the importance of coherent science instruction, in which learning opportunities are connected and contextualized by meaningful phenomena, focus on a small set of core ideas over time, and generate a need-to-know about new ideas through a set of connected lessons. Yet, this type of instruction remains uncommon in schools. We argue that science teacher education has the potential to play a powerful role in promoting coherent science instruction in schools, but to reach this potential, science teacher education programs themselves must be coherent. Based on existing literature and our work in an international collaboration focused on effective practices in science teacher education, we identify key features of coherent science teacher education programs and present a new model that we refer to as the Science Teacher Education Programmatic Coherence (STEP-C) model. The STEP-C model illustrates how key elements of science teacher education are situated relative to each other, potentially serving as a powerful tool for program design.

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.

Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo β-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.

Purpose: Improving quality of life (QoL) is a major goal of rehabilitation following spinal cord injury (SCI). However, people with disabilities in resource constrained contexts have limited access to rehabilitation and poorer health outcomes, including QoL. There is a paucity of qualitative research on the experiences of persons with SCI involved in rehabilitation programmes in low-middle income countries. This study aimed to assess participants' perceptions of the benefits of a 24-week SCI rehabilitation programme delivered as part of a pilot randomized controlled trial (RCT) in South Africa. Materials and methods: Sixteen participants, with chronic motor-incomplete tetraplegia, were enrolled in a two-arm pilot RCT involving robotic locomotor training, a novel technology, and standard activity-based training (Pan African Clinical Trial Registry (PACTR201608001647143)). Data were collected via in-depth interviews and analysed using thematic analysis. Results: Participants described several improvements in QoL, including enhanced functional independence; reduced secondary complications; and improved psychosocial and emotional well-being. Conclusions: The holistic approach to rehabilitation calls for the involvement of individuals' views about what matters to them to inform clinical practice and to highlight the role that physical activity and the perceived successes play in shaping the lived experiences after SCI. Trial registration: Pan African Clinical Trial Registry (PACTR201608001647143), registration date (21st May 2016), study start date (30th Nov 2016) IMPLICATIONS FOR REHABILITATION: Rehabilitation plays an integral role in prompting and integrating positive experiences and changes in QoL for people with spinal cord injury (SCI), especially in a resource constrained context where there is limited opportunity to participate in rehabilitation interventions. Locomotor training and activity-based training can enhance perceived functional independence and psychosocial well-being following SCI. Rather than focus on traditional physiological outcomes, rehabilitation interventions can address quality of life outcomes in order to improve well-being in a way that is meaningful to people with SCI. [ABSTRACT FROM AUTHOR]

Activity-based Training (ABT) represents the current standard of neurological rehabilitation. Robotic Locomotor Training (RLT), an innovative technique, aims to enhance rehabilitation outcomes. This study aimed to conduct a randomized pilot and feasibility trial of a locomotor training program within South Africa. Individuals with chronic traumatic motor incomplete tetraplegia (n = 16). Each intervention involved 60-minute sessions, 3x per week, for 24-weeks. Outcomes included feasibility measures and functional capacity. 17 out of 110 individuals initiated the program (recruitment rate = 15.4%) and 16 completed the program (drop-out rate = 5.8%) and attended sessions (attendance rate = 93.9%). Both groups showed a significant increase in upper extremity motor score (MS) and abdominal strength post intervention. Only the RLT group showed a significant change in lower extremity MS, with a mean increase of 3.00 [0.00; 16.5] points over time. Distance walked in the Functional Ambulatory Inventory (SCI-FAI) increased significantly (p = 0.02) over time only for the RLT group. Feasibility rates of the intervention and functional outcomes justify a subsequent powered RCT comparing RLT to ABT as an effective rehabilitation tool for potentially improving functional strength and walking capacity in people with incomplete SCI. Spinal cord injury causes severe limitations to functional capacity, independence, and quality of life. Robotic Locomotor Training is growing rehabilitation modality for people with spinal cord injury, but currently its effects on functional capacity are limited. In a relatively small sample, this study shows that large, randomized control trials are feasible within a low-income setting. The preliminary findings of this study show that 12 weeks of locomotor training can improve ambulatory function and functional strength in individuals with spinal cord injury. [ABSTRACT FROM AUTHOR]

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (∼40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ( KD < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in E. coli. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems.

Although guidelines are available for hereditary hemochromatosis, a high percentage of the recommendations within them are not shared between the different guidelines. Our main aim is to provide an objective, simple, brief, and practical set of recommendations about therapeutic aspects of HFE hemochromatosis for p.Cys282Tyr (C282Y/C282Y) homozygous genotype, based on the published scientific studies and guidelines, in a form that is reasonably comprehensible to patients and people without medical training. This final version was approved at the Hemochromatosis International meeting on 12th May 2017 in Los Angeles.

PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone and a distal member of the metallo-β-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here, we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+, and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a wide spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal-binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.