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.

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.

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.

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.

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.

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.

Part 3. Modeling of Multipollutant Profiles and Spatially Varying Health Effects with Applications to Indicators of Adverse Birth Outcomes.

The highly intercorrelated nature of air pollutants makes it difficult to examine their combined effects on health. As such, epidemiological studies have traditionally focused on single-pollutant models that use regression-based techniques to examine the marginal association between a pollutant and a health outcome. These relatively simple, additive models are useful for discerning the effect of a single pollutant on a health outcome with all other pollutants held to fixed values. However, pollutants occur in complex mixtures consisting of highly correlated combinations of individual exposures. For example, evidence for synergy among pollutants in causing health effects has been recently reviewed by Mauderly and Samet (2009). Also, studies cited in the Ozone Criteria Document (U.S. Environmental Protection Agency [U.S. EPA*] 2006) confirmed that synergisms between ozone and other pollutants have been demonstrated in laboratory studies involving humans and animals. Thus, the highly correlated nature of air pollution exposures makes marginal, single-pollutant models inadequate. This issue was raised in a report by the National Research Council (NRC 2004), which called for a multipollutant approach to air quality management. Here we present and apply a series of statistical approaches that treat patterns of covariates as a whole unit, stochastically grouping pollutant patterns into clusters and then using these cluster assignments as random effects in a regression model. Using this approach, the effect of a multipollutant pattern, or profile, is determined in a manner that takes into account the uncertainty in the clustering process. The models are set in a Bayesian framework, and in general, Markov chain Monte Carlo (MCMC) techniques (Gilks et al. 1998). For interpretation purposes, a best clustering is derived, and the uncertainty related to this best clustering is determined by utilizing model averaging techniques, in a manner such that consistent clustering obtained by the estimation process generally yields smaller standard errors while inconsistent clustering is generally associated with larger errors. These multivariate methods are applied to a range of different problems related to air pollution exposures, namely an association of multipollutant profiles with indicators of poverty and to an assessment of the association between measures of various air pollutants, patterns of socioeconomic status (SES), and birth outcomes. All of these studies involve an examination of regional-level exposures, at the census tract (CT) and census block group (CBG) levels, and individual-level outcomes throughout Los Angeles (LA) County. Results indicate that effects of pollutants vary spatially and vary in a complex interconnected manner that cannot be discerned using standard additive line ar models. Results obtaine d from these studies can be used to efficiently use limited resources to inform policies in targeting are as where air pollution reductions result in maximum health benefits.

Piwi-Interacting RNAs (piRNAs) Are Dysregulated in Renal Cell Carcinoma and Associated with Tumor Metastasis and Cancer-Specific Survival.

Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the up-regulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.

Modeling spatial effects of PM(2.5) on term low birth weight in Los Angeles County.

Air pollution epidemiological studies suggest that elevated exposure to fine particulate matter (PM2.5) is associated with higher prevalence of term low birth weight (TLBW). Previous studies have generally assumed the exposure-response of PM2.5 on TLBW to be the same throughout a large geographical area. Health effects related to PM2.5 exposures, however, may not be uniformly distributed spatially, creating a need for studies that explicitly investigate the spatial distribution of the exposure-response relationship between individual-level exposure to PM2.5 and TLBW. Here, we examine the overall and spatially varying exposure-response relationship between PM2.5 and TLBW throughout urban Los Angeles (LA) County, California. We estimated PM2.5 from a combination of land use regression (LUR), aerosol optical depth from remote sensing, and atmospheric modeling techniques. Exposures were assigned to LA County individual pregnancies identified from electronic birth certificates between the years 1995-2006 (N=1,359,284) provided by the California Department of Public Health. We used a single pollutant multivariate logistic regression model, with multilevel spatially structured and unstructured random effects set in a Bayesian framework to estimate global and spatially varying pollutant effects on TLBW at the census tract level. Overall, increased PM2.5 level was associated with higher prevalence of TLBW county-wide. The spatial random effects model, however, demonstrated that the exposure-response for PM2.5 and TLBW was not uniform across urban LA County. Rather, the magnitude and certainty of the exposure-response estimates for PM2.5 on log odds of TLBW were greatest in the urban core of Central and Southern LA County census tracts. These results suggest that the effects may be spatially patterned, and that simply estimating global pollutant effects obscures disparities suggested by spatial patterns of effects. Studies that incorporate spatial multilevel modeling with random coefficients allow us to identify areas where air pollutant effects on adverse birth outcomes may be most severe and policies to further reduce air pollution might be most effective.

Molecular basis for improved gene silencing by Dicer substrate interfering RNA compared with other siRNA variants.

The canonical exogenous trigger of RNA interference (RNAi) in mammals is small interfering RNA (siRNA). One promising application of RNAi is siRNA-based therapeutics, and therefore the optimization of siRNA efficacy is an important consideration. To reduce unfavorable properties of canonical 21mer siRNAs, structural and chemical variations to canonical siRNA have been reported. Several of these siRNA variants demonstrate increased potency in downstream readout-based assays, but the molecular mechanism underlying the increased potency is not clear. Here, we tested the performance of canonical siRNAs and several sequence-matched variants in parallel in gene silencing, RNA-induced silencing complex (RISC) assembly, stability and Argonaute (Ago) loading assays. The commonly used 19mer with two deoxythymidine overhangs (19merTT) variant performed similarly to canonical 21mer siRNA. A shorter 16mer variant (16merTT) did not perform comparably in our assays. Dicer substrate interfering RNA (dsiRNA) demonstrated better gene silencing by the guide strand (target complementary strand), better RISC assembly, persistence of the guide strand and relatively more loading of the guide strand into Ago. Hence, we demonstrate the advantageous properties of dsiRNAs at upstream, intermediate and downstream molecular steps of the RNAi pathway.

Epigenetic stability, adaptability, and reversibility in human embryonic stem cells.

The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500-2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation.

Breast cancer cell-secreted miR-199b-5p hijacks neurometabolic coupling to promote brain metastasis.

Breast cancer metastasis to the brain is a clinical challenge rising in prevalence. However, the underlying mechanisms, especially how cancer cells adapt a distant brain niche to facilitate colonization, remain poorly understood. A unique metabolic feature of the brain is the coupling between neurons and astrocytes through glutamate, glutamine, and lactate. Here we show that extracellular vesicles from breast cancer cells with a high potential to develop brain metastases carry high levels of miR-199b-5p, which shows higher levels in the blood of breast cancer patients with brain metastases comparing to those with metastatic cancer in other organs. miR-199b-5p targets solute carrier transporters (SLC1A2/EAAT2 in astrocytes and SLC38A2/SNAT2 and SLC16A7/MCT2 in neurons) to hijack the neuron-astrocyte metabolic coupling, leading to extracellular retention of these metabolites and promoting cancer cell growth. Our findings reveal a mechanism through which cancer cells of a non-brain origin reprogram neural metabolism to fuel brain metastases.

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.

Genomic mapping of 5-hydroxymethylcytosine in the human brain.

Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the sixth DNA base have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have used an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in DNA from human brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is particularly enriched at promoters and in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels for promoters with intermediate or high CpG content. However, the presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was the presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide an overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification.

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.

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.

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.

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.

De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer.

BACKGROUND: MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome. METHODS: The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers. RESULTS: More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients. CONCLUSIONS: Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.