Design principles for riboswitch function

Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence–function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands

The Genetics of Adaptation for Eight Microvirid Bacteriophages

Theories of adaptive molecular evolution have recently experienced significant expansion, and their predictions and assumptions have begun to be subjected to rigorous empirical testing. However, these theories focus largely on predicting the first event in adaptive evolution, the fixation of a single beneficial mutation. To address long-term adaptation it is necessary to include new assumptions, but empirical data are needed for guidance. To empirically characterize the general properties of adaptive walks, eight recently isolated relatives of the single-stranded DNA (ssDNA) bacteriophage φX174 (family Microviridae) were adapted to identical selective conditions. Three of the eight genotypes were adapted in replicate, for a total of 11 adaptive walks. We measured fitness improvement and identified the genetic changes underlying the observed adaptation. Nearly all phages were evolvable; nine of the 11 lineages showed a significant increase in fitness. However, fitness plateaued quickly, and adaptation was achieved through only three substitutions on average. Parallel evolution was rampant, both across replicates of the same genotype as well as across different genotypes, yet adaptation of replicates never proceeded through the exact same set of mutations. Despite this, final fitnesses did not vary significantly among replicates. Final fitnesses did vary significantly across genotypes but not across phylogenetic groupings of genotypes. A positive correlation was found between the number of substitutions in an adaptive walk and the magnitude of fitness improvement, but no correlation was found between starting and ending fitness. These results provide an empirical framework for future adaptation theory

Integrating innovations:a qualitative analysis of referral non-completion among rapid diagnostic test-positive patients in Uganda's human African trypanosomiasis elimination programme

BACKGROUND: The recent development of rapid diagnostic tests (RDTs) for human African trypanosomiasis (HAT) enables elimination programmes to decentralise serological screening services to frontline health facilities. However, patients must still undertake multiple onwards referral steps to either be confirmed or discounted as cases. Accurate surveillance thus relies not only on the performance of diagnostic technologies but also on referral support structures and patient decisions. This study explored why some RDT-positive suspects failed to complete the diagnostic referral process in West Nile, Uganda. METHODS: Between August 2013 and June 2015, 85% (295/346) people who screened RDT-positive were examined by microscopy at least once; 10 cases were detected. We interviewed 20 RDT-positive suspects who had not completed referral (16 who had not presented for their first microscopy examination, and 4 who had not returned for a second to dismiss them as cases after receiving discordant [RDT-positive, but microscopy-negative results]). Interviews were analysed thematically to examine experiences of each step of the referral process. RESULTS: Poor provider communication about HAT RDT results helped explain non-completion of referrals in our sample. Most patients were unaware they were tested for HAT until receiving results, and some did not know they had screened positive. While HAT testing and treatment is free, anticipated costs for transportation and ancillary health services fees deterred many. Most expected a positive RDT result would lead to HAT treatment. RDT results that failed to provide a definitive diagnosis without further testing led some to question the expertise of health workers. For the four individuals who missed their second examination, complying with repeat referral requests was less attractive when no alternative diagnostic advice or treatment was given. CONCLUSIONS: An RDT-based surveillance strategy that relies on referral through all levels of the health system is inevitably subject to its limitations. In Uganda, a key structural weakness was poor provider communication about the possibility of discordant HAT test results, which is the most common outcome for serological RDT suspects in a HAT elimination programme. Patient misunderstanding of referral rationale risks harming trust in the whole system and should be addressed in elimination programmes

cDNA Immunization of Mice with Human Thyroglobulin Generates Both Humoral and T Cell Responses: A Novel Model of Thyroid Autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis

Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3

The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the “split-SET” domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20

Growth Hormone Promotes Hair Cell Regeneration in the Zebrafish (Danio rerio) Inner Ear following Acoustic Trauma

BACKGROUND: Previous microarray analysis showed that growth hormone (GH) was significantly upregulated following acoustic trauma in the zebrafish (Danio rerio) ear suggesting that GH may play an important role in the process of auditory hair cell regeneration. Our objective was to examine the effects of exogenous and endogenous GH on zebrafish inner ear epithelia following acoustic trauma. METHODOLOGY/PRINCIPAL FINDINGS: We induced auditory hair cell damage by exposing zebrafish to acoustic overstimulation. Fish were then injected intraperitoneally with either carp GH or buffer, and placed in a recovery tank for either one or two days. Phalloidin-, bromodeoxyuridine (BrdU)-, and TUNEL-labeling were used to examine hair cell densities, cell proliferation, and apoptosis, respectively. Two days post-trauma, saccular hair cell densities in GH-treated fish were similar to that of baseline controls, whereas buffer-injected fish showed significantly reduced densities of hair cell bundles. Cell proliferation was greater and apoptosis reduced in the saccules, lagenae, and utricles of GH-treated fish one day following trauma compared to controls. Fluorescent in situ hybridization (FISH) was used to examine the localization of GH mRNA in the zebrafish ear. At one day post-trauma, GH mRNA expression appeared to be localized perinuclearly around erythrocytes in the blood vessels of the inner ear epithelia. In order to examine the effects of endogenous GH on the process of cell proliferation in the ear, a GH antagonist was injected into zebrafish immediately following acoustic trauma, resulting in significantly decreased cell proliferation one day post-trauma in all three zebrafish inner ear end organs. CONCLUSIONS/SIGNIFICANCE: Our results show that exogenous GH promotes post-trauma auditory hair cell regeneration in the zebrafish ear through stimulating proliferation and suppressing apoptosis, and that endogenous GH signals are present in the zebrafish ear during the process of auditory hair cell regeneration

Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3

Centromeres are characterized by the centromere-specific H3 variant CENP-A, which is embedded in chromatin with a pattern characteristic of active transcription that is required for centromere identity. It is unclear how centromeres remain transcriptionally active despite being flanked by repressive pericentric heterochromatin. To further understand centrochromatin’s response to repressive signals, we nucleated a Polycomb-like chromatin state within the centromere of a human artificial chromosome (HAC) by tethering the methyltransferase EZH2. This led to deposition of the H3K27me3 mark and PRC1 repressor binding. Surprisingly, this state did not abolish HAC centromere function or transcription, and this apparent resistance was not observed on a noncentromeric locus, where transcription was silenced. Directly tethering the reader/repressor PRC1 bypassed this resistance, inactivating the centromere. We observed analogous responses when tethering the heterochromatin Editor Suv39h1-methyltransferase domain (centromere resistance) or reader HP1α (centromere inactivation), respectively. Our results reveal that the HAC centromere can resist repressive pathways driven by H3K9me3/H3K27me3 and may help to explain how centromeres are able to resist inactivation by flanking heterochromatin