Binding-Induced Molecular Amplifier as a Universal Detection Platform for Biomolecules and Biomolecular Interaction.

Techniques that detect multiple classes of biomolecules and biomolecular interactions from biological or patient samples are highly desirable for applications ranging from accurate disease diagnosis to deciphering comprehensive biological processes. Because of the large variations in target recognition, signal transduction, and instrumentation, it is technically challenging to generalize a single detection method to a diverse range of analytical targets. Herein, we introduce a binding-induced molecular amplifier (BIMA) strategy that translates a variety of biomolecules and biomolecular interactions into unified predesigned DNA barcode in homogeneous solutions. On the basis of a three-dimensional DNA-walking mechanism, BIMA not only translates various targets into a unified barcode but also amplifies the translation by generating multiple barcode molecules in response to a single input target molecule. Using this strategy, we have successfully expanded the uses of a simple toehold-mediated strand displacement beacon for the sensitive detection of multiple classes of targets, including nucleic acids, proteins, and protein-protein interactions.

Ethnocultural influences in how people prefer to obtain and receive health information.

AIMS AND OBJECTIVES: To develop an understanding of south Asian and Chinese people's preferences about where to find health information and how best to receive health information, relative to their white counterparts. BACKGROUND: South Asian and Chinese ethnic groups represent the largest proportion of Canada's growing visible minorities. There may be challenges to ensuring that south Asian and Chinese people have access to health information in the same way that others do. DESIGN: Qualitative descriptive. METHODS: Fifty-two participants (12 white, 16 south Asian and 24 Chinese) engaged in six focus groups (two for each ethnocultural group). Focus groups were conducted in English, Punjabi and Cantonese, with the assistance of Punjabi and Cantonese interpreters. Questions were focused on how participants have preferred or would prefer to receive health information (e.g., when, where, what format, from whom), as well as the facilitators and barriers to understanding the health information. RESULTS: Participants agreed that although physicians were their primary source for health information, they also used written materials, media and the Internet to glean information. Participants identified concerns regarding the use of technical jargon by healthcare providers. South Asians and Chinese referred to their English language fluency and the lack of ethnoculturally specific information as additional challenges to understanding information they were offered. Whether and how family members were included in the communication process, also varied by ethnocultural group. CONCLUSIONS: As Canada welcomes immigrants from other countries, and its population becomes more diverse, healthcare providers need to have an understanding of the potential diversity in how to approach offering health information. RELEVANCE TO CLINICAL PRACTICE: Healthcare providers need to consider what people of different ethnocultural backgrounds need when developing effective health communication strategies.

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression.

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression.

An H4K16 histone acetyltransferase mediates decondensation of the X chromosome in C. elegans males.

BACKGROUND: In C. elegans, in order to equalize gene expression between the sexes and balance X and autosomal expression, two steps are believed to be required. First, an unknown mechanism is hypothesized to upregulate the X chromosome in both sexes. This mechanism balances the X to autosomal expression in males, but creates X overexpression in hermaphrodites. Therefore, to restore the balance, hermaphrodites downregulate gene expression twofold on both X chromosomes. While many studies have focused on X chromosome downregulation, the mechanism of X upregulation is not known. RESULTS: To gain more insight into X upregulation, we studied the effects of chromatin condensation and histone acetylation on gene expression levels in male C. elegans. We have found that the H4K16 histone acetyltransferase MYS-1/Tip60 mediates dramatic decondensation of the male X chromosome as measured by FISH. However, RNA-seq analysis revealed that MYS-1 contributes only slightly to upregulation of gene expression on the X chromosome. These results suggest that the level of chromosome decondensation does not necessarily correlate with the degree of gene expression change in vivo. Furthermore, the X chromosome is more sensitive to MYS-1-mediated decondensation than the autosomes, despite similar levels of H4K16ac on all chromosomes, as measured by ChIP-seq. H4K16ac levels weakly correlate with gene expression levels on both the X and the autosomes, but highly expressed genes on the X chromosome do not contain exceptionally high levels of H4K16ac. CONCLUSION: These results indicate that H4K16ac and chromosome decondensation influence regulation of the male X chromosome; however, they do not fully account for the high levels of gene expression observed on the X chromosomes.

Balancing up and downregulation of the C. elegans X chromosomes.

In Caenorhabditis elegans, males have one X chromosome and hermaphrodites have two. Emerging evidence indicates that the male X is transcriptionally more active than autosomes to balance the single X to two sets of autosomes. Because upregulation is not limited to males, hermaphrodites need to strike back and downregulate expression from the two X chromosomes to balance gene expression in their genome. Hermaphrodite-specific downregulation involves binding of the dosage compensation complex to both Xs. Advances in recent years revealed that the action of the dosage compensation complex results in compaction of the X chromosomes, changes in the distribution of histone modifications, and ultimately limiting RNA Polymerase II loading to achieve chromosome-wide gene repression.

The C. elegans dosage compensation complex mediates interphase X chromosome compaction.

BACKGROUND: Dosage compensation is a specialized gene regulatory mechanism which equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC localizes to both X chromosomes in hermaphrodites to downregulate gene expression by half. The DCC contains a subcomplex (condensin I(DC)) similar to the evolutionarily conserved condensin complexes which play fundamental roles in chromosome dynamics during mitosis and meiosis. Therefore, mechanisms related to mitotic chromosome condensation have been long hypothesized to mediate dosage compensation. However experimental evidence was lacking. RESULTS: Using 3D FISH microscopy to measure the volumes of X and chromosome I territories and to measure distances between individual loci, we show that hermaphrodite worms deficient in DCC proteins have enlarged interphase X chromosomes when compared to wild type. By contrast, chromosome I is unaffected. Interestingly, hermaphrodite worms depleted of condensin I or II show no phenotype. Therefore X chromosome compaction is specific to condensin I(DC). In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory. CONCLUSION: These results support the idea that condensin I(DC), and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction. Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.

Pluripotent cells will not dosage compensate.

Dosage compensation is the mechanism that balances gene expression levels between males and females as well as between the X chromosome and autosomes. In mammals, loss of pluripotency and differentiation are closely linked with the onset of dosage compensation. Pluripotency factors negatively regulate Xist (the non-coding RNA that triggers X chromosome inactivation) and positively regulate Tsix, a repressor of Xist, to inhibit dosage compensation. In addition, X chromosome dose also regulates exit from the pluripotent state. A double dose of X chromosomes in undifferentiated female cells inhibits the MAPK and Gsk3 signaling pathways and activates the Akt pathway, thereby blocking differentiation. Here we review our recent report, which showed that the onset of dosage compensation is also linked to the loss of pluripotency in C. elegans. We discuss these findings in light of what is known about pluripotency and differentiation in this organism.

Condensin-mediated chromosome organization and gene regulation.

In many organisms sexual fate is determined by a chromosome-based method which entails a difference in sex chromosome-linked gene dosage. Consequently, a gene regulatory mechanism called dosage compensation equalizes X-linked gene expression between the sexes. Dosage compensation initiates as cells transition from pluripotency to differentiation. In Caenorhabditis elegans, dosage compensation is achieved by the dosage compensation complex (DCC) binding to both X chromosomes in hermaphrodites to downregulate gene expression by twofold. The DCC contains a subcomplex (condensin I(DC)) similar to the evolutionarily conserved condensin complexes which play a fundamental role in chromosome dynamics during mitosis. Therefore, mechanisms related to mitotic chromosome condensation are hypothesized to mediate dosage compensation. Consistent with this hypothesis, monomethylation of histone H4 lysine 20 is increased, whereas acetylation of histone H4 lysine 16 is decreased, both on mitotic chromosomes and on interphase dosage compensated X chromosomes in worms. These observations suggest that interphase dosage compensated X chromosomes maintain some characteristics associated with condensed mitotic chromosome. This chromosome state is stably propagated from one cell generation to the next. In this review we will speculate on how the biochemical activities of condensin can achieve both mitotic chromosome compaction and gene repression.

Activation of the Ca(2+)-sensing receptor increases renal claudin-14 expression and urinary Ca(2+) excretion.

Kidney stones are a prevalent clinical condition imposing a large economic burden on the healthcare system. Hypercalciuria remains the major risk factor for development of a Ca(2+)-containing stone. The kidney's ability to alter Ca(2+) excretion in response to changes in serum Ca(2+) is in part mediated by the Ca(2+)-sensing receptor (CaSR). Recent studies revealed renal claudin-14 (Cldn14) expression localized to the thick ascending limb (TAL) and its expression to be regulated via the CaSR. We find that Cldn14 expression is increased by high dietary Ca(2+) intake and by elevated serum Ca(2+) levels induced by prolonged 1,25-dihydroxyvitamin D3 administration. Consistent with this, activation of the CaSR in vivo via administration of the calcimimetic cinacalcet hydrochloride led to a 40-fold increase in Cldn14 mRNA. Moreover, overexpression of Cldn14 in two separate cell culture models decreased paracellular Ca(2+) flux by preferentially decreasing cation permeability, thereby increasing transepithelial resistance. These data support the existence of a mechanism whereby activation of the CaSR in the TAL increases Cldn14 expression, which in turn blocks the paracellular reabsorption of Ca(2+). This molecular mechanism likely facilitates renal Ca(2+) losses in response to elevated serum Ca(2+). Moreover, dysregulation of the newly described CaSR-Cldn14 axis likely contributes to the development of hypercalciuria and kidney stones.