Regulation of Enhancers by SUMOylation Through TFAP2C Binding and Recruitment of HDAC Complex to the Chromatin.

Enhancers are fundamental to gene regulation. Post-translational modifications by the small ubiquitin-like modifiers (SUMO) modify chromatin regulation enzymes, including histone acetylases and deacetylases. However, it remains unclear whether SUMOylation regulates enhancer marks, acetylation at the 27th lysine residue of the histone H3 protein (H3K27Ac). To investigate whether SUMOylation regulates H3K27Ac, we performed genome-wide ChIP-seq analyses and discovered that knockdown (KD) of the SUMO activating enzyme catalytic subunit UBA2 reduced H3K27Ac at most enhancers. Bioinformatic analysis revealed that TFAP2C-binding sites are enriched in enhancers whose H3K27Ac was reduced by UBA2 KD. ChIP-seq analysis in combination with molecular biological methods showed that TFAP2C binding to enhancers increased upon UBA2 KD or inhibition of SUMOylation by a small molecule SUMOylation inhibitor. However, this is not due to the SUMOylation of TFAP2C itself. Proteomics analysis of TFAP2C interactome on the chromatin identified histone deacetylation (HDAC) and RNA splicing machineries that contain many SUMOylation targets. TFAP2C KD reduced HDAC1 binding to chromatin and increased H3K27Ac marks at enhancer regions, suggesting that TFAP2C is important in recruiting HDAC machinery. Taken together, our findings provide insights into the regulation of enhancer marks by SUMOylation and TFAP2C and suggest that SUMOylation of proteins in the HDAC machinery regulates their recruitments to enhancers.

Crystal Structure and Enzymology of Solanum tuberosum Inositol Tris/Tetrakisphosphate Kinase 1 (StITPK1).

Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates.

Mechanism of primer synthesis by Primase-Polymerases.

Members of the primase-polymerase (Prim-Pol) superfamily are found in all domains of life and play diverse roles in genome stability, including primer synthesis during DNA replication, lesion repair and damage tolerance. This review focuses primarily on Prim-Pol members capable of de novo primer synthesis that have experimentally derived structural models available. We discuss the mechanism of DNA primer synthesis initiation by Prim-Pol catalytic domains, based on recent structural and functional studies. We also describe a general model for primer initiation that also includes the ancillary domains/subunits, which stimulate the initiation of primer synthesis.

Reverse transcriptases prime DNA synthesis.

The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.

Reverse transcriptases (RTs) are replicative enzymes that copy RNA into DNA and undertake roles, including viral replication, retrotransposition and telomere maintenance. The initiation of RT synthesis activities is usually dependent on the presence of a primer. The current dogma proposes that a variety of indirect, RT-independent, priming mechanisms instigate synthesis. However, this study establishes that CRISPR-associated RTs (CARTs) are capable of priming DNA synthesis from scratch, which enables the capture of foreign genetic material for storage in CRISPR arrays. The authors also report that other notable RT family members, including retrotransposon RTs, telomerase and retroviral RT are, surprisingly, able to directly catalyze primer synthesis. These findings significantly alter our understanding of priming mechanisms utilised by RTs in various biological pathways.

Long non-coding RNA lncMGC mediates the expression of TGF-ß-induced genes in renal cells via nucleosome remodelers.

Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-ß (TGF-ß), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling. Methods: In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-ß, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined. Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation-qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-ß-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-ß-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-ß-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene. Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells.

Barriers in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) management: perspectives from health practitioners.

OBJECTIVES: Chronic prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a complex condition which causes a significant burden on the diagnosed individuals. Assessment and management are perplexing, often resulting in unsatisfactory outcomes. Existing research has only focused on patients' perspectives of pain experiences, but scant evidence is available to understand the barriers that undermine effective pain management. Using an exploratory approach, this study examined these barriers from practitioners' perspectives. METHODS: Twelve semi-structured interviews were conducted with practitioners across disciplines who have experience in chronic pelvic pain management in males. Practitioners expressed their views and experiences in supporting men with CP/CPPS and what barriers they perceived when providing treatment for patients. Data were analysed using reflexive thematic analysis supported by NVivo software. RESULTS: Five broad and interrelated themes were identified: (1) Where to Start, (2) Insufficient Resources, (3) Prioritisation, (4) Training and Confident Practice and (5) Constraints in Help-Seeking. CONCLUSIONS: Practitioners value multimodal management using a biopsychosocial approach; however, practical challenges prevent practitioners from choosing and applying this approach in clinical practice. The findings also identified some unique challenges faced by men with CP/CPPS consistent with previous evidence from patient perspective. Refining terminology, developing specific resources, and increasing psychosocial treatment options are urgently needed.

Precision sirolimus dosing in children: The potential for model-informed dosing and novel drug monitoring.

The mTOR inhibitor sirolimus is prescribed to treat children with varying diseases, ranging from vascular anomalies to sporadic lymphangioleiomyomatosis to transplantation (solid organ or hematopoietic cell). Precision dosing of sirolimus using therapeutic drug monitoring (TDM) of sirolimus concentrations in whole blood drawn at the trough (before the next dose) time-point is the current standard of care. For sirolimus, trough concentrations are only modestly correlated with the area under the curve, with R 2 values ranging from 0.52 to 0.84. Thus, it should not be surprising, even with the use of sirolimus TDM, that patients treated with sirolimus have variable pharmacokinetics, toxicity, and effectiveness. Model-informed precision dosing (MIPD) will be beneficial and should be implemented. The data do not suggest dried blood spots point-of-care sampling of sirolimus concentrations for precision dosing of sirolimus. Future research on precision dosing of sirolimus should focus on pharmacogenomic and pharmacometabolomic tools to predict sirolimus pharmacokinetics and wearables for point-of-care quantitation and MIPD.

Psychological management of patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS): a systematic review.

OBJECTIVES: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a complex condition. Despite recommendations for the inclusion of non-pharmacological treatment in the management of CP/CPPS, the focus has predominantly been on the inclusion of physical therapies with minimal discussion of psychological interventions. Therefore, this systematic review aimed to evaluate peer-reviewed studies of psychological interventions for men with CP/CPPS to determine their therapeutic efficacy and quality of intervention. METHODS: The review was registered in PROSPERO and based on PRISMA 2020 protocol. The systematic literature search was conducted in six databases. Quantitative studies of psychological intervention for adult men with CP/CPPS that provided outcome measures of pain, quality of life and/or psychological symptoms were reviewed. The Oxford level of evidence and Quality Assessment Tool for Quantitative Studies developed by the Effective Public Health Practice were employed. RESULTS: A total of 4,503 studies were reviewed; seven met the inclusion criteria. The included studies were randomised controlled trials, cohort, repeated measures, and case-series studies, with most including combined treatment for CP/CPPS. Cognitive therapy, cognitive behavioural therapy, or paradoxical relaxation training were found to be effective. However, high risks of bias were found in all included studies, limiting the generalisability and reliability of findings. CONCLUSIONS: Evidence is preliminary but shows promise for psychological treatment either as a combined or standalone treatment for CP/CPPS. However, there is a need to develop research with a more rigorous methodology to evaluate psychological treatments for men with CP/CPPS.

Improving epinephrine autoinjector usability and carriage frequency among patients at risk of anaphylaxis: a quality improvement initiative.

Although epinephrine autoinjectors (EAIs) are crucial for the management of anaphylaxis, patient carriage frequency of EAI is as low as 57% and usage of EAIs is erroneous 35%-43% of the time. Our objective was to improve patient carrying frequency of EAI and understanding of EAI usage.We implemented a quality improvement initiative using consistent closed-loop education, redesigned clinic workflow, electronic medical record reminder-based interventions, and educational materials to improve patient EAI carriage compliance and understanding of EAI indications and proper technique.The percentage of our patients who carried the EAI at all times increased from 55% to 93% in 6 months. Participants knowledge of EAI indications also improved from 22% to 91%. Patient demonstration scores of the EAI device improved from 21% to 91% as well.Our quality improvement interventions demonstrated a significant improvement>80% in EAI carriage frequency, knowledge of indications, and proper device technique.

Molecular basis for the initiation of DNA primer synthesis.

During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.

CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation.

CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

The situation in Hong Kong during the COVID-19 pandemic.

This article briefly discusses the reactions and responses toward the COVID-19 pandemic in Hong Kong, amid major political strife. The traumatic memories from severe acute respiratory syndrome in 2003 as well as the displease and distrust toward the Hong Kong government have aggravated mental health distress during the pandemic while this adversity has also increased community support. This article discusses the specific challenges of the COVID-19 pandemic in Hong Kong and proposes lessons learned during the pandemic, including strategies for mental health professionals and the community to reduce psychological distress. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Surgeon and patient perceptions of cure in advanced gastrointestinal malignancies: Are we on the same page?

BACKGROUND AND OBJECTIVES: Effective communication is essential to complex shared decision making and is associated with improved recovery and pain control. However, patients and surgeons often have disparate expectations of treatment efficacy and perceptions of cure for advanced malignancies. This study measures correlation of patient and surgeon expectations with perceptions of cure. METHODS: Our prospective study surveying surgeon-patient dyads before and after surgical consultation was performed for advanced abdominal malignancy between July and November 2017 at a single NCI designated cancer center using electronic questionnaires. RESULTS: Patients and surgeons' own opinions regarding surgical candidacy (Q1), chance at cure (Q2), and life expectancy (Q3) did not measurably change from pre- to postvisit survey as evidenced by unchanged response concordance (patients Q1 P = .82; Q2 P = .81; and Q3 P = .53; surgeon responses Q1: P = .17; Q2: P = .32; and Q3: P = .50). Patient and surgeon perception of likelihood of cure and of estimated life expectancy remained discordant in pre- and postvisit surveys (Q2: P = .006 and Q3: P = .03). CONCLUSIONS: These data highlight the stark differences between patient and surgeon perceptions of cure and prognosis of gastrointestinal cancers. These results prove that a larger scale study using this electronic questionnaire is feasible and important to better understand these differences and enhance shared decision making.

The impact of financial toxicity in gastrointestinal cancer patients.

BACKGROUND: Biopsychosocial distress screening is a critical component of comprehensive cancer care. Financial issues are a common source of distress in this patient population. This study uses a biopsychosocial distress screening tool to determine the factors associated with financial toxicity and the impact of these stressors on gastrointestinal cancer patients. METHODS: A 48-question, proprietary distress screening tool was administered to patients with gastrointestinal malignancies from 2009 to 2015. This validated, electronically-administered tool is given to all new patients. Responses were recorded on a 5-point Likert scale from 1 (not a problem) to 5 (very severe problem), with responses rated at ≥3 indicative of distress. Univariate and multivariate logistic regressions were used to analyze the data. RESULTS: Most of the 1,027 patients had colorectal (50%) or hepatobiliary (31%) malignancies. Additionally, 34% of all patients expressed a high level of financial toxicity. Age greater than 65 (odds ratio: 0.63, 95% confidence interval: 0.47-0.86, P < .01), college education (odds ratio: 0.53, 95% confidence interval: 0.38-0.73, P < .0001), being partnered (odds ratio: 0.61, 95% confidence interval: 0.44-0.84, P < .01), and annual income greater than $40,000 (odds ratio: 0.27, 95% confidence interval: 0.19-0.38, P < .0001) were all protective against financial toxicity on univariate analysis. Also, heavy tobacco use was associated significantly with increased distress on univariate analysis (odds ratio: 2.79, 95% confidence interval: 1.38-5.78, P < .01). With the exception of partnered status (odds ratio: 1.18, 95% confidence interval: 0.76-1.85, P = .46), all these variables retained their significant association with financial toxicity in the multivariate model. CONCLUSION: Financial toxicity impacts a large number of cancer patients. Further study of at-risk populations may identify patients who would benefit from pre-emptive education and counseling interventions as part of their routine cancer care.

Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism.

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

Reprogramming of DNA methylation at NEUROD2-bound sequences during cortical neuron differentiation.

The characteristics of DNA methylation changes that occur during neurogenesis in vivo remain unknown. We used whole-genome bisulfite sequencing to quantitate DNA cytosine modifications in differentiating neurons and their progenitors isolated from mouse brain at the peak of embryonic neurogenesis. Localized DNA hypomethylation was much more common than hypermethylation and often occurred at putative enhancers within genes that were upregulated in neurons and encoded proteins crucial for neuronal differentiation. The hypomethylated regions strongly overlapped with mapped binding sites of the key neuronal transcription factor NEUROD2. The 5-methylcytosine oxidase ten-eleven translocation 2 (TET2) interacted with NEUROD2, and its reaction product 5-hydroxymethylcytosine accumulated at the demethylated regions. NEUROD2-targeted differentially methylated regions retained higher methylation levels in Neurod2 knockout mice, and inducible expression of NEUROD2 caused TET2-associated demethylation at its in vivo binding sites. The data suggest that the reorganization of DNA methylation in developing neurons involves NEUROD2 and TET2-mediated DNA demethylation.

Socioeconomic Status and the Prediction of Health Promoting Dietary Behaviours: A Systematic Review and Meta-Analysis Based on the Theory of Planned Behaviour.

BACKGROUND: Dietary behaviours are strongly patterned by socioeconomic status (SES). However, the role of SES in the self-regulation of health promoting dietary behaviours is not fully understood. This systematic review with meta-analysis investigated whether four individual-level measures of SES (income, occupation, education, and ethnicity) moderate the relationships between Theory of Planned Behaviour (TPB) variables and different health promoting dietary behaviours in adults. METHODS: A systematic literature search identified 106 studies from 77 articles providing information on TPB variables, SES, and health promoting dietary behaviours-choosing health promoting foods and restricting health compromising foods. Random-effects meta-analyses were conducted to generate pooled correlations corrected for sampling and measurement error, and meta-regression was used to test moderating effects of study-level SES. RESULTS: All TPB variables were significantly and positively associated with both health promoting dietary behaviours, with intention having the strongest correlation with behaviour. However, none of the relationships between TPB variables and health promoting dietary behaviours were significantly moderated by study-level SES. CONCLUSIONS: Results suggest robust associations between TPB predictors and healthy dietary behaviour that are not patterned by individual-level SES measures.

Identification of Tissue-Specific DNA Methylation Signatures for Thyroid Nodule Diagnostics.

PURPOSE: Thyroid cancer is frequently difficult to diagnose due to an overlap of cytologic features between malignant and benign nodules. This overlap leads to unnecessary removal of the thyroid in patients without cancer. While providing some improvement over cytopathologic diagnostics, molecular methods frequently fail to provide a correct diagnosis for thyroid nodules. These approaches are based on the difference between cancer and adjacent thyroid tissue and assume that adjacent tissues are the same as benign nodules. However, in contrast to adjacent tissues, benign thyroid nodules can contain genetic alterations that can be found in cancer.Experimental Design: For the development of a new molecular diagnostic test for thyroid cancer, we evaluated DNA methylation in 109 thyroid tissues by using genome-wide single-base resolution DNA methylation analysis. The test was validated in a retrospective cohort containing 65 thyroid nodules. RESULTS: By conducting reduced representation bisulfite sequencing in 109 thyroid specimens, we found significant differences between adjacent tissue, benign nodules, and cancer. These tissue-specific signatures are strongly linked to active enhancers and cancer-associated genes. Based on these signatures, we developed a new epigenetic approach for thyroid diagnostics. According to the validation cohort, our test has an estimated specificity of 97% [95% confidence interval (CI), 81-100], sensitivity of 100% (95% CI, 87-100), positive predictive value of 97% (95% CI, 83-100), and negative predictive value of 100% (95% CI, 86-100). CONCLUSIONS: These data show that epigenetic testing can provide outstanding diagnostic accuracy for thyroid nodules.See related commentary by Mitmaker et al., p. 457.

Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts.

BACKGROUND: Bone is one of the most frequent metastatic sites of advanced breast cancer. Current therapeutic agents aim to inhibit osteoclast-mediated bone resorption but only have palliative effects. During normal bone remodeling, the balance between bone resorption and osteoblast-mediated bone formation is essential for bone homeostasis. One major function of osteoblast during bone formation is to secrete type I procollagen, which will then be processed before being crosslinked and deposited into the bone matrix. METHODS: Small RNA sequencing and quantitative real-time PCR were used to detect miRNA levels in patient blood samples and in the cell lysates as well as extracellular vesicles of parental and bone-tropic MDA-MB-231 breast cancer cells. The effects of cancer cell-derived extracellular vesicles isolated by ultracentrifugation and carrying varying levels of miR-218 were examined in osteoblasts by quantitative real-time PCR, Western blot analysis, and P1NP bone formation marker analysis. Cancer cells overexpressing miR-218 were examined by transcriptome profiling through RNA sequencing to identify intrinsic genes and pathways influenced by miR-218. RESULTS: We show that circulating miR-218 is associated with breast cancer bone metastasis. Cancer-secreted miR-218 directly downregulates type I collagen in osteoblasts, whereas intracellular miR-218 in breast cancer cells regulates the expression of inhibin ß subunits. Increased cancer secretion of inhibin ßA results in elevated Timp3 expression in osteoblasts and the subsequent repression of procollagen processing during osteoblast differentiation. CONCLUSIONS: Here we identify a twofold function of cancer-derived miR-218, whose levels in the blood are associated with breast cancer metastasis to the bone, in the regulation of type I collagen deposition by osteoblasts. The adaptation of the bone niche mediated by miR-218 might further tilt the balance towards osteolysis, thereby facilitating other mechanisms to promote bone metastasis.

High type I error and misrepresentations in search for transgenerational epigenetic inheritance: response to Guerrero-Bosagna.

In a recent paper, we described our efforts in search for evidence supporting epigenetic transgenerational inheritance caused by endocrine disrupter chemicals. One aspect of our study was to compare genome-wide DNA methylation changes in the vinclozolin-exposed fetal male germ cells (n = 3) to control samples (n = 3), their counterparts in the next, unexposed, generation (n = 3 + 3) and also in adult spermatozoa (n = 2 + 2) in both generations. We reported finding zero common hits in the intersection of these four comparisons. In our interpretation, this result did not support the notion that DNA methylation provides a mechanism for a vinclozolin-induced transgenerational male infertility phenotype. In response to criticism by Guerrero-Bosagna regarding our statistical power in the above study, here we provide power calculations to clarify the statistical power of our study and to show the validity of our conclusions. We also explain here how our data is misinterpreted in the commentary by Guerrero-Bosagna by leaving out important data points from consideration.Please see related Correspondence article: xxx (13059_2016_982) and related Research article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0619-z.