Nuclear localization of Argonaute 2 is affected by cell density and may relieve repression by microRNAs.

Argonaute protein is associated with post-transcriptional control of cytoplasmic gene expression through miRNA-induced silencing complexes (miRISC). Specific cellular and environmental conditions can trigger AGO protein to accumulate in the nucleus. Localization of AGO is central to understanding miRNA action, yet the consequences of AGO being in the nucleus are undefined. We show nuclear enrichment of AGO2 in HCT116 cells grown in two-dimensional culture to high density, HCT116 cells grown in three-dimensional tumor spheroid culture, and human colon tumors. The shift in localization of AGO2 from cytoplasm to nucleus de-represses cytoplasmic AGO2-eCLIP targets that were candidates for canonical regulation by miRISC. Constitutive nuclear localization of AGO2 using an engineered nuclear localization signal increases cell migration. Critical RNAi factors also affect the localization of AGO2. Knocking out an enzyme essential for miRNA biogenesis, DROSHA, depletes mature miRNAs and restricts AGO2 localization to the cytoplasm, while knocking out the miRISC scaffolding protein, TNRC6, results in nuclear localization of AGO2. These data suggest that AGO2 localization and miRNA activity can be regulated depending on environmental conditions, expression of mature miRNAs, and expression of miRISC cofactors. Localization and expression of core miRISC protein machinery should be considered when investigating the roles of miRNAs.

RNAi in cell nuclei: potential for a new layer of biological regulation and a new strategy for therapeutic discovery.

RNA interference is almost always associated with post-transcriptional silencing in the cytoplasm. MicroRNAs (miRNAs) and critical RNAi protein factors like argonaute (AGO) and trinucleotide repeat binding containing 6 protein (TNRC6), however, are also found in cell nuclei, suggesting that nuclear miRNAs may be targets for gene regulation. Designed small duplex RNAs (dsRNAs) can modulate nuclear processes such as transcription and splicing, suggesting that they can also provide leads for therapeutic discovery. The goal of this Perspective is to provide the background on nuclear RNAi necessary to guide discussions on whether nuclear RNAi can play a role in therapeutic development programs.

Consequences of depleting TNRC6, AGO, and DROSHA proteins on expression of microRNAs.

The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and trinucleotide repeat containing six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRBase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The MirGeneDB resource offers the closest alignment with experimental results. In contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA-dependence, and physical association with AGO when forming hypotheses related to their function.

Argonaute binding within human nuclear RNA and its impact on alternative splicing.

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

Argonaute binding within 3'-untranslated regions poorly predicts gene repression.

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3'-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3'-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

Implementing a Skin-to-Skin Care and Parent Touch Initiative in a Tertiary Cardiac and Surgical Neonatal Intensive Care Unit.

BACKGROUND: Skin-to-skin care (SSC) has been integrated as an essential component of developmental care for preterm infants. Despite documented benefits, SSC is not routinely practiced in the cardiac and surgical neonatal intensive care unit, with a predominantly term population, due to staff apprehension, patient factors and acuity, and environmental constraints. PURPOSE: The purpose of this quality improvement project was to increase SSC, parental holds, and parent touch events for infants in our cardiac and surgical neonatal intensive care unit. When traditional SSC was not possible, alternative holds and alternative parent touch (APT) methods were encouraged. METHODS: Quality improvement and qualitative descriptive methodology were utilized to assess baseline, develop education and practice changes, and evaluate the use of SSC, holds, and APT methods at 12 and 18 months postintervention. Implementation included educational tools and resource development, simulations, peer champions, in-class teaching, and team huddles. Decisions around the type of hold and parent touch were fluid and reflected complex infant, family, staff, and physical space needs. FINDINGS: Given its initial scarcity, there was an increased frequency of SSC and variety of holds or APT events. Staff survey results indicated support for the practice and outlined persistent barriers. IMPLICATIONS FOR PRACTICE: Skin-to-skin care, holds, and APT practices are feasible and safe for term and preterm infants receiving highly instrumented and complex cardiac and surgical care. IMPLICATIONS FOR RESEARCH: Future research regarding the intervention's impact on neurodevelopmental outcomes of infants and on parent resilience in the surgical and cardiac neonatal intensive care unit is warranted.

Implementation of the Neonatal Nurse Practitioner Role in a Community Hospital's Labor, Delivery, and Level 1 Postpartum Unit.

BACKGROUND: A level 1 community hospital with a labor, delivery, recovery, and postpartum (LDRP) unit delivering over 2800 babies per year was operating without dedicated neonatal resuscitation and stabilization support. PURPOSE: With lack of funding and space to provide an onsite level 2 neonatal intensive care unit (NICU), a position was created to provide neonatal nurse practitioner (NNP) coverage to support the LDRP unit. METHOD: The article describes the innovative solution of having an NNP team rotate from a regional neonatal intensive care program to a busy community LDRP unit. The presence of the NNP supported the development and integration of the advanced practice nursing role with interdisciplinary team members in both the LDRP and the emergency department. RESULTS: The NNP was able to provide expertise, leadership, and mentorship for neonatal resuscitation and stabilization as well as education and consultation on neonatal care. In addition to the services provided by the NNP for infant's requiring acute care, the NNP provided transitional support for those infants who remained with their mothers in the LDRP unit. Furthermore, time required by the neonatal transport team to stabilize babies before transport to the NICU was decreased with NNP presence. IMPLICATIONS FOR PRACTICE: The divergence from practice of the traditional NNP clinical role in the NICU setting to more of a consultant and nursing leader has proven to be a valued role at the community hospital. IMPLICATIONS FOR RESEARCH: A solid economic analysis of the cost-effectiveness of the NNP role in this community hospital is warranted.

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

BACKGROUND: Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. METHODS: This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. RESULTS: Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. CONCLUSIONS: Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated.

Development of a LAG-3 immunohistochemistry assay for melanoma.

AIMS: A robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumour tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardised, well-characterised assay to measure its expression. This study aims to describe LAG-3 scoring criteria and present the specificity, sensitivity, analytical precision and reproducibility of this assay. METHODS: The specificity of the assay was investigated by antigen competition and with LAG3 knockout cell lines. A melanin pigment removal procedure was implemented to prevent melanin interference in IHC interpretation. Formalin-fixed paraffin-embedded (FFPE) human melanoma samples with a range of LAG-3 expression levels were used to assess the sensitivity and analytical precision of the assay with a ≥1% cut-off to determine LAG-3 positivity. Interobserver and intraobserver reproducibility were evaluated with 60 samples in intralaboratory studies and 70 samples in interlaboratory studies. RESULTS: The LAG-3 IHC method demonstrated performance suitable for analysis of LAG-3 IC expression in clinical melanoma samples. The pretreatment step effectively removed melanin pigment that could interfere with interpretation. LAG-3 antigen competition and analysis of LAG3 knockout cell lines indicated that the 17B4 antibody clone binds specifically to LAG-3. The intrarun repeatability, interday, interinstrument, interoperator and inter-reagent lot reproducibility demonstrated a high scoring concordance (>95%). The interobserver and intraobserver reproducibility and overall interlaboratory and intralaboratory reproducibility also showed high scoring concordance (>90%). CONCLUSIONS: We have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC.

Nuclear Localization of Argonaute is affected by Cell Density and May Relieve Repression by microRNAs.

Argonaute protein is associated with post-transcriptional control of cytoplasmic gene expression through miRNA-induced silencing complexes (miRISC). Specific cellular and environmental conditions can trigger AGO protein to accumulate in the nucleus. Localization of AGO is central to understanding miRNA action, yet the consequences of AGO being in the nucleus are undefined. We show nuclear enrichment of AGO2 in HCT116 cells grown in two-dimensional culture to high density, HCT116 cells grown in three-dimensional tumor spheroid culture, and human colon tumors. The shift in localization of AGO2 from cytoplasm to nucleus de-represses cytoplasmic AGO2-eCLIP targets that were candidates for canonical regulation by miRISC. Constitutive nuclear localization of AGO2 using an engineered nuclear localization signal increases cell migration. Critical RNAi factors also affect the localization of AGO2. Knocking out an enzyme essential for miRNA biogenesis, DROSHA, depletes mature miRNAs and restricts AGO2 localization to the cytoplasm, while knocking out the miRISC scaffolding protein, TNRC6, results in nuclear localization of AGO2. These data suggest that AGO2 localization and miRNA activity can be regulated depending on environmental conditions, expression of mature miRNAs, and expression of miRISC cofactors. Localization and expression of core miRISC protein machinery should be considered when investigating the roles of miRNAs.

Oxygen use for preterm infants: factors that may influence clinical decisions surrounding oxygen titration.

Preterm infants in neonatal intensive care units frequently require oxygen therapy. Clinicians are responsible for titrating oxygen to maximize the benefits and minimize the risks of this therapy. Studies have identified various toxic effects of oxygen on the developing tissues of the preterm infant; however, optimal target SpO(2) ranges have not been identified. Current trends in neonatology are focusing on defining optimal oxygen saturation ranges to improve infant outcomes and to decrease complications associated with the oxygen use. Consequently, research-based guidelines are being developed in neonatal intensive care units to guide oxygen administration. As target oxygen saturation ranges are developed, issues regarding health care professional compliance with these ranges have been identified. The specific reasons for this noncompliance have not been widely explored. However, factors such as nursing shortages, staffing issues, and a de-emphasis on staff education surrounding oxygen use have been offered as possible reasons. Understanding factors shaping clinical decision-making about oxygen titration is critical when designing policies and educational programs to change oxygen titration practice and ultimately improve patient outcomes. In this article, the literature outlining the importance of oxygen titration for preterm infants is reviewed. Discussion then focuses on factors that influence clinical decision-making and how these factors may influence decisions surrounding the use of oxygen for preterm infants.

Patterns of GRP78 and MTJ1 expression in primary cutaneous malignant melanoma.

Cell surface expression of glucose-regulated protein 78 (GRP78) occurs in several types of cancer; however, its role in the behavior of primary cutaneous melanoma is not well studied. The association of cell surface GRP78 with other proteins such as MTJ1 stimulates cell proliferation. In this study, we characterized the pattern of expression of GRP78 and MTJ1 in invasive primary cutaneous melanomas and analyzed the relationships between the pattern of expression and various clinicopathological parameters. We found two patterns of GRP78 expression in invasive primary cutaneous melanoma. One pattern showed a gradual fading of protein expression from superficial to deeper levels within the same tumor. The second pattern of expression showed a similar fading with an abrupt regaining of expression at the deep invasive edge of the melanoma. These two distinct patterns of GRP78 expression correlated with both patient survival and depth of tumor invasion. A moderate MTJ1 expression was found to be associated with decreased patient survival; however, no significant associations were observed between patterns of GRP78 and MTJ1 expression. Our study (1) describes two distinct patterns of GRP78 in invasive primary cutaneous melanoma, (2) inversely correlates regain of GRP78 expression with patient survival, and (3) suggests a modifying effect of MTJ1 on GRP78 in enhancing tumor aggressiveness.

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription.

Cellular dielectric spectroscopy: a label-free comprehensive platform for functional evaluation of endogenous receptors.

The CellKey (MDS Sciex, South San Francisco, CA) system enables comprehensive pharmacological evaluation of cell surface receptors, including G-protein coupled receptors (GPCRs) and tyrosine kinase receptors, using adherent and suspension cell lines and primary cells. A unique application enabled by the ability of the CellKey system to reliably quantify activation of endogenous receptors is receptor panning. This application allows investigators to easily screen disease-relevant cell types for functionally active target receptors by treating cells with a panel of receptor-specific ligands. Receptor panning of multiple cell types including Chinese hamster ovary, human embryonic kidney 293, HeLa, U-937, U-2 OS, and TE671 cells resulted in the identification of many functionally active, differently coupled endogenous GPCRs, some of which have not been previously documented in the literature. Upon detecting GPCR activation in live cells, unique cellular dielectric spectroscopy (CDS) response profiles are generated within minutes that reflect the signaling pathways utilized and have been shown to be characteristic of Gs, Gq, and Gi GPCRs. The fact that the CDS response profiles are predictive of the G-protein coupling mechanism of the receptor was demonstrated by using examples of subtype-selective agonists/antagonists to identify the subtypes of the endogenous histamine and beta-adrenergic receptors expressed in U-2 OS cells. A direct correlation is shown between receptor subtype G-protein coupling and CDS response profile. In addition, complex pharmacology, including detection of partial agonism and Schild analysis for endogenous receptors, is presented. The CellKey system allows investigators to conduct studies using endogenously expressed receptors to generate data that are physiologically relevant and in disease context.

Evidence-Based Cancer Survivorship Activities for Comprehensive Cancer Control.

INTRODUCTION: One of six priorities of CDC's National Comprehensive Cancer Control Program (NCCCP) is to address the needs of cancer survivors within the local population served by individually funded states, tribes, and territories. This report examines cancer survivorship activities implemented in five NCCCP grantees, which have initiated evidence-based activities outlined in A National Action Plan for Cancer Survivorship: Advancing Public Health Strategies (NAP). METHODS: NCCCP action plans, submitted annually to CDC, from 2010 to 2014 were reviewed in February 2015 to assess implementation of cancer survivorship activities and recommended strategies consistent with the NAP. Four state-level and one tribal grantee with specific activities related to one of each of the four NAP strategies were chosen for inclusion. Brief case reports describing the initiation and impact of implemented activities were developed in collaboration with each grantee program director. RESULTS: New Mexico, South Carolina, Vermont, Washington state, and Fond Du Lac Band of Lake Superior Chippewa programs each implemented activities in surveillance and applied research; communication, education, and training; programs, policies, and infrastructure; and access to quality care and services. CONCLUSIONS: This report provides examples for incorporating cancer survivorship activities within Comprehensive Cancer Control programs of various sizes, demographic makeup, and resource capacity. New Mexico, South Carolina, Vermont, Washington state, and Fond Du Lac Band developed creative cancer survivorship activities that meet CDC recommendations. NCCCP grantees can follow these examples by implementing evidence-based survivorship interventions that meet the needs of their specific populations.

Array-based karyotyping in plasma cell neoplasia after plasma cell enrichment increases detection of genomic aberrations.

The discovery of genomic abnormalities present in monoclonal plasma cells has diagnostic, prognostic, and disease-monitoring implications in plasma cell neoplasms (PCNs). However, technical and disease-related limitations hamper the detection of these abnormalities using cytogenetic analysis or fluorescence in situ hybridization (FISH). In this study, 28 bone marrow specimens with known PCNs were examined for the presence of genomic abnormalities using microarray analysis after plasma cell enrichment. Cytogenetic analysis was performed on 15 of 28 samples, revealing disease-related genomic aberrations in only 3 (20%) of 15 cases. FISH analysis was performed on enriched plasma cells and detected aberrations in 84.6% of specimens while array comparative genomic hybridization (aCGH) detected abnormalities in 89.3% of cases. Furthermore, aCGH revealed additional abnormalities in 24 cases compared with FISH alone. We conclude that aCGH after plasma cell enrichment, in combination with FISH, is a valuable approach for routine clinical use in achieving a more complete genetic characterization of patients with PCN.

Detection of genomic abnormalities in multiple myeloma: the application of FISH analysis in combination with various plasma cell enrichment techniques.

Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells (PCs) that accumulate in the bone marrow. A number of different genomic abnormalities are associated with MM; however, detection of these by fluorescence in situ hybridization (FISH) can be limited by the percentage of PCs in the specimen. In this study, we tested 20 bone marrow specimens with known MM and a low concentration of monoclonal PCs for the presence of genomic abnormalities using FISH in combination with various PC enrichment techniques: magnetic cell sorting, targeted manual scoring, and automated image analysis. In addition, flow cytometric cell sorting of PCs in combination with FISH analysis was also tested for minimal residual disease applications. Different parameters were evaluated when assessing the detection efficiency of each approach. FISH results are highly dependent on the chosen enrichment method. We describe the evaluation of different techniques applicable for various laboratory settings and specimen parameters.