Prediction of DNA i-motifs via machine learning.

i-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. The programme iM-Seeker incorporates a Balanced Random Forest classifier trained on genome-wide iMab antibody-based CUT&Tag sequencing data to predict the folding status and an Extreme Gradient Boosting regressor to estimate the folding strength according to both literature biophysical data and our in-house biophysical experiments. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination (R2) of 0.642 on the test set. Model interpretation confirms that the nucleotide composition of the C-rich sequence significantly affects iM stability, with a positive correlation with sequences containing cytosine and thymine and a negative correlation with guanine and adenine.

Photoresponsive Emulsion-Templated Porous Materials via Orthogonal Photoclick Chemistry.

The functionalization of emulsion-templated porous polymers (polyHIPEs) utilizing modern and efficient chemistries is an important avenue for tailoring the properties of these scaffolds for specific and specialized applications. Herein, tetrazole photoclick chemistry is utilized for the efficient functionalization of polyHIPEs synthesized from various monomer systems and polymerization chemistries. Using both radical polymerization and thiol-ene polymerization, polyHIPEs with well-defined, interconnected open-cell morphologies are synthesized with tetrazole concentrations ranging from 0 to 5 w/v %, with the pore diameters ranging from 3 to 24 µm. Analyzed by fluorescence spectroscopy, FTIR spectroscopy, and confocal microscopy, spatially controlled functionalization to generate photopatterned fluorescent polyHIPEs is demonstrated via the reaction with residual acrylate and thiol groups. In addition, the scaffolds can be readily functionalized with external dipolarophiles such as acrylates to incorporate a functionality onto the polyHIPE surface. With many functional tetrazoles also reported in the literature, a PEG-tetrazole is also used to explore the photoinduced functionalization of polyHIPEs possessing tunable ratios of thiol and acrylate groups, and the effect on fluorescence, wettability, and biocompatibility is analyzed. Overall, the reaction is shown to be a broadly applicable tool for polyHIPE functionalization with many avenues for further development toward specific applications.

Epigenetic modification of cytosines fine tunes the stability of i-motif DNA.

i-Motifs are widely used in nanotechnology, play a part in gene regulation and have been detected in human nuclei. As these structures are composed of cytosine, they are potential sites for epigenetic modification. In addition to 5-methyl- and 5-hydroxymethylcytosine modifications, recent evidence has suggested biological roles for 5-formylcytosine and 5-carboxylcytosine. Herein the human telomeric i-motif sequence was used to examine how these four epigenetic modifications alter the thermal and pH stability of i-motifs. Changes in melting temperature and transitional pH depended on both the type of modification and its position within the i-motif forming sequence. The cytosines most sensitive to modification were next to the first and third loops within the structure. Using previously described i-motif forming sequences, we screened the MCF-7 and MCF-10A methylomes to map 5-methylcytosine and found the majority of sequences were differentially methylated in MCF7 (cancerous) and MCF10A (non-cancerous) cell lines. Furthermore, i-motif forming sequences stable at neutral pH were significantly more likely to be epigenetically modified than traditional acidic i-motif forming sequences. This work has implications not only in the epigenetic regulation of DNA, but also allows discreet tunability of i-motif stability for nanotechnological applications.

Substitution of Cytosine with Guanylurea Decreases the Stability of i-Motif DNA.

Both 5-aza-2'-deoxycytidine (decitabine) and its primary breakdown product, 2'-deoxyriboguanylurea (GuaUre-dR), have been shown to act as mutagens and epimutagens that cause replication stress and alter both DNA methylation and gene expression patterns. As cytosine analogues, both are expected to be preferentially incorporated into regions of GC skew where runs of cytosine residues are sequestered on one strand and guanine residues on the other. Given that such regions have been identified as sites with the potential for effects on gene expression and replication stress linked to formation of alternative DNA secondary structures, it is of interest to determine the influence that these base analogues might have on the stability of structures of this kind. Here we report that incorporation of GuaUre-dR into an i-motif-forming sequence decreases both the thermal and pH stability of an i-motif despite the apparent ability of GuaUre-dR to base pair with cytosine.

Identification of multiple genomic DNA sequences which form i-motif structures at neutral pH.

i-Motifs are alternative DNA secondary structures formed in cytosine-rich sequences. Particular examples of these structures, traditionally assumed to be stable only at acidic pH, have been found to form under near-physiological conditions. To determine the potential impact of these structures on physiological processes, investigation of sequences with the capacity to fold under physiological conditions is required. Here we describe a systematic study of cytosine-rich DNA sequences, with varying numbers of consecutive cytosines, to gain insights into i-motif DNA sequence and structure stability. i-Motif formation was assessed using ultraviolet spectroscopy, circular dichroism and native gel electrophoresis. We found that increasing cytosine tract lengths resulted in increased thermal stability; sequences with at least five cytosines per tract folded into i-motif at room temperature and neutral pH. Using these results, we postulated a folding rule for i-motif formation, analogous to (but different from) that for G-quadruplexes. This indicated that thousands of cytosine-rich sequences in the human genome may fold into i-motif structures under physiological conditions. Many of these were found in locations where structure formation is likely to influence gene expression. Characterization of a selection of these identified i-motif forming sequences uncovered 17 genomic i-motif forming sequence examples which were stable at neutral pH.

Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences.

There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

Reversible DNA i-motif to hairpin switching induced by copper(II) cations.

i-Motif DNA structures have previously been utilised for many different nanotechnological applications, but all have used changes in pH to fold the DNA. Herein we describe how copper(II) cations can alter the conformation of i-motif DNA into an alternative hairpin structure which is reversible by chelation with EDTA.

Metal complex interactions with DNA.

Increasing numbers of DNA structures are being revealed using biophysical, spectroscopic and genomic methods. The diversity of transition metal complexes is also growing, as the unique contributions that transition metals bring to the overall structure of metal complexes depend on the various coordination numbers, geometries, physiologically relevant redox potentials, as well as kinetic and thermodynamic characteristics. The vast range of ligands that can be utilised must also be considered. Given this diversity, a variety of biological interactions is not unexpected. Specifically, interactions with negatively-charged DNA can arise due to covalent/coordinate or subtle non-coordinate interactions such as electrostatic attraction, groove binding and intercalation as well as combinations of all of these modes. The potential of metal complexes as therapeutic agents is but one aspect of their utility. Complexes, both new and old, are currently being utilised in conjunction with spectroscopic and biological techniques to probe the interactions of DNA and its many structural forms. Here we present a review of metal complex-DNA interactions in which several binding modes and DNA structural forms are explored.

Deep imaging: how much of the proteome does current top-down technology already resolve?

Effective proteome analyses are based on interplay between resolution and detection. It had been claimed that resolution was the main factor limiting the use of two-dimensional gel electrophoresis. Improved protein detection now indicates that this is unlikely to be the case. Using a highly refined protocol, the rat brain proteome was extracted, resolved, and detected. In order to overcome the stain saturation threshold, high abundance protein species were excised from the gel following standard imaging. Gels were then imaged again using longer exposure times, enabling detection of lower abundance, less intensely stained protein species. This resulted in a significant enhancement in the detection of resolved proteins, and a slightly modified digestion protocol enabled effective identification by standard mass spectrometric methods. The data indicate that the resolution required for comprehensive proteome analyses is already available, can assess multiple samples in parallel, and preserve critical information concerning post-translational modifications. Further optimization of staining and detection methods promises additional improvements to this economical, widely accessible and effective top-down approach to proteome analysis.

Top-down proteomics: enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection.

The large-scale resolution and detection of proteins from complex native mixtures is fundamental to quantitative proteomic analyses. Comprehensive analyses depend on careful tissue handling and quantitative protein extraction and assessment. To most effectively link these analyses with an understanding of underlying molecular mechanisms, it is critical that all protein types - isoforms, splice variants and those with functionally important PTMs - are quantitatively extracted with high reproducibility. Methodological details concerning protein extraction and resolution using 2DE are discussed with reference to current in-gel protein detection limits. We confirm a significant increase in total protein, and establish that extraction, resolution and detection of phospho- and glycoproteins are improved following automated frozen disruption relative to manual homogenisation. The quality of 2DE protein resolution is established using third-dimension separations and 'deep imaging'; substantially more proteins/protein species than previously realised are actually resolved by 2DE. Thus, the key issue for effective proteome analyses is most likely to be detection, not resolution. Thus, these systematic methodological and technical advances further solidify the role of 2DE in top-down proteomics. By routinely assessing as much proteomic data from a sample as possible, 2DE enables more detailed and critical insights into molecular mechanisms underlying different physiological states.

A Systems Biology Approach to Understanding the Mechanisms of Action of an Alternative Anticancer Compound in Comparison to Cisplatin.

Many clinically available anticancer compounds are designed to target DNA. This commonality of action often yields overlapping cellular response mechanisms and can thus detract from drug efficacy. New compounds are required to overcome resistance mechanisms that effectively neutralise compounds like cisplatin and those with similar chemical structures. Studies have shown that 56MESS is a novel compound which, unlike cisplatin, does not covalently bind to DNA, but is more toxic to many cell lines and active against cisplatin-resistant cells. Furthermore, a transcriptional study of 56MESS in yeast has implicated iron and copper metabolism as well as the general yeast stress response following challenge with 56MESS. Beyond this, the cytotoxicity of 56MESS remains largely uncharacterised. Here, yeast was used as a model system to facilitate a systems-level comparison between 56MESS and cisplatin. Preliminary experiments indicated that higher concentrations than seen in similar studies be used. Although a DNA interaction with 56MESS had been theorized, this work indicated that an effect on protein synthesis/ degradation was also implicated in the mechanism(s) of action of this novel anticancer compound. In contrast to cisplatin, the different mechanisms of action that are indicated for 56MESS suggest that this compound could overcome cisplatin resistance either as a stand-alone treatment or a synergistic component of therapeutics.

Mandatory reporting of child abuse and neglect: crafting a positive process for health professionals and caregivers.

Health professionals working with children and their families are often required by law to report to governmental authorities any reasonable suspicion of child abuse and/or neglect. Extant research has pointed toward various barriers to reporting, with scant attention to positive processes to support the reporting process. This paper focuses on the context for mandatory reporting and evidence-informed practice for supporting a more structured and purposeful process of mandatory reporting. These practical strategies discusses: (1) the factors that positively influence the relationship between a child's caregivers and the mandated health professional reporter; (2) a framework and specific skills for discussing concerns about maltreatment and reporting to child protective services with the caregiver(s); and (3) the need for further training and education of health professionals.

Comparative gait analysis of ankle arthrodesis and arthroplasty: initial findings of a prospective study.

BACKGROUND: Little is known about functional outcomes of ankle arthroplasty compared with arthrodesis. This study compared pre-surgical and post-surgical gait measures in both patient groups. METHODS: Eighteen patients with end-stage ankle arthritis participated in an ongoing longitudinal study (pre-surgery, 12 months post-surgery) involving gait analysis, assessment of pain and physical function. Outcome measures included temporal-distance, kinematic and kinetic data, the Short Form 36 (SF-36) body pain score, and average daily step count. A mixed effects linear model was used to detect effects of surgical group (arthrodesis and arthroplasty, n = 9 each) with walking speed as a covariate (α = 0.05). RESULTS: Both groups were similar in demographics and anthropometrics. Followup time was the same for each group. There were no complications in either group. Pain decreased (p < 0.001) and gait function improved (gait velocity, p = 0.02; stride length, p = 0.035) in both groups. Neither group increased average daily step count. Joint range of motion (ROM) differences were observed between groups after surgery (increased hip ROM in arthrodesis, p = 0.001; increased ankle ROM in arthroplasty, p = 0.036). Peak plantar flexor moment increased in arthrodesis patients and decreased in arthroplasty patients (p = 0.042). CONCLUSION: Initial findings of this ongoing clinical study indicate pain reduction and improved gait function 12 months after surgery for both treatments. Arthroplasty appears to regain more natural ankle joint function, with increased ROM. Long-term follow up should may reveal more clinically meaningful differences.

Long-term alterations to the brain transcriptome in a maternal voluntary consumption model of fetal alcohol spectrum disorders.

Many women continue to consume low to moderate quantities of alcohol during pregnancy, which can result in the variable neurobehavioural effects in the absence of physiological abnormalities that characterize fetal alcohol spectrum disorders (FASD). Previously, we reported that a mouse model for FASD based on voluntary maternal ethanol consumption throughout gestation resulted in offspring that showed mild developmental delay, anxiety-related traits, and deficits in spatial learning. Here, we extend this model by evaluating the gene expression changes that occur in the adult brain of C57BL/6J mice prenatally exposed to ethanol via maternal preference drinking. The results of two independent expression array experiments indicate that ethanol induces subtle but consistent changes to global gene expression. Gene enrichment analysis showed over-represented gene ontology classifications of cellular, embryonic, and nervous system development. Molecular network analysis supported these classifications, with significant networks related to cellular and tissue development, free radical scavenging, and small molecule metabolism. Further, a number of genes identified have previously been implicated in FASD-relevant neurobehavioural phenotypes such as cognitive function (Ache, Bcl2, Cul4b, Dkc1, Ebp, Lcat, Nsdh1, Sstr3), anxiety (Bcl2), attention deficit hyperactivity disorder (Nsdh1), and mood disorders (Bcl2, Otx2, Sstr3). The results suggest a complex residual "footprint" of neurodevelopmental ethanol exposure that may provide a new perspective for identifying mechanisms that underlie the life-long persistence of FASD-related cognitive and behavioural alterations, including potential targets for treatment.

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods.

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

Maternal voluntary drinking in C57BL/6J mice: advancing a model for fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) remain the most common preventable cause of behavioural abnormalities and cognitive deficits, yet little is known about the biological mechanisms involved in FASD pathology. Maternal voluntary ethanol consumption in mice may be a useful model for establishing the biological basis of moderate ethanol exposure phenotypes, which make up the majority of FASD cases. We have employed a two-bottle choice paradigm of maternal ethanol consumption throughout gestation and the early postnatal period in C57BL/6J mice. We assessed the efficacy of this model to produce a range of FASD-relevant phenotypes and evaluated gene expression changes in the adult offspring. Results showed stable maternal consumption and lack of maternal care differences between ethanol-consuming and water-only dams. Ethanol-exposed offspring showed delays in neonatal reflex and coordination development. Further, ethanol-exposed adolescent mice showed decreased activity in a novel environment that appeared to be the result of novelty-induced anxiety, and acquisition learning deficits. Evaluation of the neurotransmitter-associated genes Gabra6, Glra1, and Grin2c revealed significant down-regulation of Glra1 and Grin2c in the brains of ethanol-exposed young adult males. These results suggest that this model is able to produce a range of behavioural phenotypes consistent with prenatal ethanol exposure and may be used to evaluate resulting long-term genetic changes. Given the range of genetic resources available for inbred mouse strains, the model described here may prove to be a useful tool in evaluating the molecular basis of FASD.

Effects of plant-derived polyphenols on TNF-alpha and nitric oxide production induced by advanced glycation endproducts.

Advanced glycation endproducts (AGEs) accumulate on protein deposits including the beta-amyloid plaques in Alzheimer's disease. AGEs interact with the "receptor for advanced glycation endproducts", and transmit their signals using intracellular reactive oxygen species as second messengers. Ultimately, AGEs induce the expression of a variety of pro-inflammatory markers including the tumor necrosis factor (TNF-alpha) and inducible nitric oxide (NO) synthase. Antioxidants that act intracellularly, including polyphenols, have been shown to scavenge these "signaling" reactive oxygen species, and thus perform in an anti-inflammatory capacity. This study tested the pure compounds apigenin and diosmetin as well as extracts from silymarin, uva ursi (bearberry) and green olive leaf for their ability to attenuate AGE-induced NO and TNF-alpha production. All five tested samples inhibited BSA-AGE-induced NO production in a dose-dependent manner. Apigenin and diosmetin were most potent, and exhibited EC(50) values approximately 10 microM. In contrast, TNF-alpha expression was only reduced by apigenin, diosmetin and silymarin; not by the bearberry and green olive leaf extracts. In addition, the silymarin and bearberry extracts caused significant cell death at concentrations >or=10 microg/mL and >or=50 microg/mL, respectively. In conclusion, we suggest that plant-derived polyphenols might offer therapeutic opportunities to delay the progression of AGE-mediated and receptor for advanced glycation endproducts-mediated neuro-inflammatory diseases including Alzheimer's disease.