RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination.

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome.

The Relation Between the Paraphilias and Anxiety in Men: A Case-Control Study.

Despite a multitude of theoretical views, it is still unclear how individuals develop and sustain paraphilic interests (e.g., sexual attraction to children, interest in non-consensual violence). It is also not clear from these views why many paraphilic interests, and especially many paraphilias and paraphilic disorders, are much more common in men than in women. One possible factor affecting male's higher rate of paraphilias is anxiety, because anxiety can potentiate sexual arousal in men. We speculated that paraphilic interests could develop when feelings of anxiety are recurrently generated by atypical sexual stimuli, and when that anxiety repeatedly potentiates sexual arousal, reinforcing sexual response to atypical stimuli. It follows that men with paraphilic interests are susceptible to anxiety disorders, because an anxiety disorder would facilitate the hypothesized developmental process. We conducted a retrospective file review of 1048 consecutive patients (944 male patients retained for analysis) referred to an outpatient sexual behavior clinic at a psychiatric hospital to investigate the link between paraphilias and anxiety. Male patients with a paraphilia had 1.64 greater odds than male patients without a paraphilia of having been diagnosed with an anxiety disorder, but they also had elevated rates of many other types of disorders. Therefore, there does not seem to be a specific link between paraphilias and anxiety in this sample. The discovery of a general link between the paraphilias and psychological disorders in men opens new avenues for studying the developmental origins and consequences of male paraphilic interests.

Mammary-like Carcinoma of the vulva: A diagnostic challenge.

Mammary-like carcinoma of the vulva is incredibly rare with less than 30 cases published since 1935, and the similarities of such pathology between breast cancer metastases, squamous adenocarcinoma, Bartholin gland carcinomas, etc, make an accurate diagnosis challenging. A diagnosis can be made utilizing immunohistochemical staining and patient history to rule out more likely causes such as metastases to ensure a correct diagnosis.

The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.

The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3' end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3' end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets.

Phenotypic plasticity in normal breast derived epithelial cells.

BACKGROUND: Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. RESULTS: All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. CONCLUSIONS: The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools.

Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72.

Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P < 0.05). C9ORF72 cases included both limb and bulbar onset disease and all cases showed combined upper and lower motor neuron degeneration (amyotrophic lateral sclerosis). Thus, clinically, C9ORF72 cases show the features of a relatively rapidly progressive, but otherwise typical, variant of amyotrophic lateral sclerosis associated with both familial and sporadic presentations. Dementia was present in the patient or a close family member in 22/62 cases with C9ORF72 mutation (35%) based on diagnoses established from retrospective clinical case note review that may underestimate significant cognitive changes in late disease. All the C9ORF72 mutation cases showed classical amyotrophic lateral sclerosis pathology with TDP-43 inclusions in spinal motor neurons. Neuronal cytoplasmic inclusions and glial inclusions positive for p62 immunostaining in non-motor regions were strongly over-represented in the C9ORF72 cases. Extra-motor pathology in the frontal cortex (P < 0.0005) and the hippocampal CA4 subfield neurons (P < 0.0005) discriminated C9ORF72 cases strongly from the rest of the cohort. Inclusions in CA4 neurons were not present in non-C9ORF72 cases, indicating that this pathology predicts mutation status.

Cutaneous meningioma: a potential diagnostic pitfall in p63 positive cutaneous neoplasms.

Cutaneous meningiomas are divided into three groups. Type I lesions present at birth and are derived from ectopic arachnoid cells. Type II lesions usually present in adults and are derived from arachnoid cells surrounding nerve bundles. Type III lesions are due to direct extension or metastasis from dural-based neoplasms. Dural-based meningiomas are known to express p63. The aim of our study is to examine the expression of p63 in type II and type III meningioma. Two cases of cutaneous meningioma (type II and type III) were evaluated for the expression of p63, EMA, CK 5/6, S100 and CD31. The cells of interest were spindled to epithelioid and arranged in a whorling pattern. Immunohistochemical staining showed expression of EMA and p63 in both cases, while stains for CK 5/6, S100 and CD31 were negative. Among cutaneous tumors, p63 is considered a marker of epithelial derivation, as it is positive in epidermal and adnexal neoplasms. It is important to be aware of p63 expression in the context of cutaneous meningioma to avoid misinterpretation as an epithelial tumor. On the basis of our small study, it is unlikely that p63 expression would be helpful in distinguishing between type II and type III meningioma, as both may be p63-positive.

Expression of MiTF may be helpful in differentiating cellular neurothekeoma from plexiform fibrohistiocytic tumor (histiocytoid predominant) in a partial biopsy specimen.

BACKGROUND: Overlapping histopathologic features of cellular neurothekeoma (CNT) and plexiform fibrohistiocytic tumor (PFHT), when both are predominantly composed of histiocytoid cells, make distinction between these entities challenging. Some have suggested that CNT and PFHT are related entities. No prior study has demonstrated a reliable immunohistochemical panel to differentiate these entities. METHODS: Skin biopsies diagnosed as CNT and PFHT, from 2004 to 2010 were retrieved with accompanying pathology reports. Each case was reviewed by at least 2 dermatopathologists and 2 soft tissue pathologists for confirmation of diagnosis. All cases were then evaluated for immunohistochemical expression of PAX2, NKIC3, CD10, and microphthalmia transcription factor (MiTF). RESULTS: Histopathologically, the histiocytoid areas of each tumor shared similar architecture, demonstrating nests and fascicles of histiocytoid to spindled cells, with some separation of nests by collagen bands. Both CNT and PFHT were uniformly positive for NKIC3 and CD10, and both were frequently PAX2 positive. MiTF was strongly and diffusely positive in CNT and was consistently negative in the PFHT. CONCLUSIONS: CNT and PFHT share many histopathologic features and immunohistochemical staining patterns. Of the stains we evaluated, we found that expression of MiTF may be a reliable marker for distinguishing CNT from histiocytoid-predominant PFHT, especially in instances where only a small part of the tumor is sampled for evaluation.

Medical genetics and epigenetics of telomerase.

Telomerase is a specialized reverse transcriptase that extends and maintains the terminal ends of chromosomes, or telomeres. Since its discovery in 1985 by Nobel Laureates Elizabeth Blackburn and Carol Greider, thousands of articles have emerged detailing its significance in telomere function and cell survival. This review provides a current assessment on the importance of telomerase regulation and relates it in terms of medical genetics. In this review, we discuss the recent findings on telomerase regulation, focusing on epigenetics and non-coding RNAs regulation of telomerase, such as microRNAs and the recently discovered telomeric-repeat containing RNA transcripts. Human genetic disorders that develop due to mutations in telomerase subunits, the role of single nucleotide polymorphisms in genes encoding telomerase components and diseases as a result of telomerase regulation going awry are also discussed. Continual investigation of the complex regulation of telomerase will further our insight into the use of controlling telomerase activity in medicine.

Extra-acral cutaneous/soft tissue sclerosing perineurioma: an under-recognized entity in the differential of CD34-positive cutaneous neoplasms.

BACKGROUND: Perineuriomas are an uncommon group of tumors composed of perineurial cells of peripheral nerve sheath lineage. Variants include soft tissue (extraneural), intraneural, sclerosing, reticular and plexiform forms. Sclerosing perineuriomas have been reported to occur almost exclusively on the hands of young men. Herein, we report three extra-acral cutaneous/soft tissue perineuriomas that show significant associated sclerosis. METHODS: Skin biopsy specimens from three patients were evaluated for cytomorphology and degree of associated sclerosis, which was classified as either focal or diffuse. Immunohistochemical expression of epithelial membrane antigen (EMA), CD34 and S-100 was also assessed to facilitate further classification. RESULTS: Histopathologically, the tumors showed both focal and diffuse sclerosis, and lesional cells were generally spindled in shape. Immunohistochemical staining showed diffuse positivity for CD34 and focal or diffuse positivity for EMA. The cells uniformly lacked expression of S-100 protein. CONCLUSIONS: Sclerosing perineuriomas can occur in extra-acral locations and should be considered in the differential of EMA-positive cutaneous spindle-cell proliferations. It is also important to recognize that perineuriomas can express CD34 and should be considered in the differential diagnosis of CD34-positive cutaneous neoplasms.

A Pivotal Response Treatment Package for Children With Autism Spectrum Disorder: An RCT.

OBJECTIVES: Our aim was to conduct a randomized controlled trial to evaluate a pivotal response treatment package (PRT-P) consisting of parent training and clinician-delivered in-home intervention on the communication skills of children with autism spectrum disorder. METHODS: Forty-eight children with autism spectrum disorder and significant language delay between 2 and 5 years old were randomly assigned to PRT-P (n = 24) or the delayed treatment group (n = 24) for 24 weeks. The effect of treatment on child communication skills was assessed via behavioral coding of parent-child interactions, standardized parent-report measures, and blinded clinician ratings. RESULTS: Analysis of child utterances during the structured laboratory observation revealed that, compared with the delayed treatment group, children in PRT-P demonstrated greater improvement in frequency of functional utterances (F1,41 = 6.07; P = .026; d = 0.61). The majority of parents in the PRT-P group (91%) were able to implement pivotal response treatment (PRT) with fidelity within 24 weeks. Children receiving PRT-P also demonstrated greater improvement on the Brief Observation of Social Communication Change, on the Clinical Global Impressions Improvement subscale, and in number of words used on a parent-report questionnaire. CONCLUSIONS: This is the first 24-week randomized controlled trial in which community treatment is compared with the combination of parent training and clinician-delivered PRT. PRT-P was effective for improving child social communication skills and for teaching parents to implement PRT. Additional research will be needed to understand the optimal combination of treatment settings, intensity, and duration, and to identify child and parent characteristics associated with treatment response.

Importance of preswing rectus femoris activity in stiff-knee gait.

Stiff-knee gait is characterized by diminished and delayed knee flexion during swing. Rectus femoris transfer surgery, a common treatment for stiff-knee gait, is often recommended when a patient exhibits prolonged activity of the rectus femoris muscle during swing. Treatment outcomes are inconsistent, in part, due to limited understanding of the biomechanical factors contributing to stiff-knee gait. This study used a combination of gait analysis and dynamic simulation to examine how activity of the rectus femoris during swing, and prior to swing, contribute to knee flexion. A group of muscle-actuated dynamic simulations was created that accurately reproduced the gait dynamics of ten subjects with stiff-knee gait. These simulations were used to examine the effects of rectus femoris activity on knee motion by eliminating rectus femoris activity during preswing and separately during early swing. The increase in peak knee flexion by eliminating rectus femoris activity during preswing (7.5+/-3.1 degrees ) was significantly greater on average (paired t-test, p=0.035) than during early swing (4.7+/-3.6 degrees ). These results suggest that preswing rectus femoris activity is at least as influential as early swing activity in limiting the knee flexion of persons with stiff-knee gait. In evaluating rectus femoris activity for treatment of stiff-knee gait, preswing as well as early swing activity should be examined.

Physician integration is back--and more important than ever.

Current affiliation strategies tend to focus on joint ventures and employment. Careful planning and organizing of a joint venture can mitigate their associated legal, tax, regulatory, and cultural risks. The success of an employment model depends upon a compensation structure that aligns the incentives of physicians and the hospital. Fora successful affiliation program, hospitals should determine needs and trends, implement strategic planning, and conduct due diligence.

Plasmacytoid Urothelial Carcinoma of the Urinary Bladder: A Clinicopathologic and Immunohistochemical Analysis of 49 Cases.

OBJECTIVES: Plasmacytoid urothelial carcinoma (PUC) of the bladder is a rare histologic variant. We retrospectively analyzed a large series of bladder PUC from a single institution. METHODS: The patients consisted of 44 men and five women with a mean age of 62 years (range, 45-86 years). RESULTS: PUC was pure in 23 cases and mixed with other histologic types in 26 cases. All PUCs diffusely invaded the bladder wall. Most PUCs lacked immunoreactivity for the retinoblastoma (RB) gene protein (12/32) and E-cadherin (8/30). Of the 44 patients with follow-up information, 25 died of PUC at a mean of 23 months, whereas 19 patients were alive at a mean of 22 months. CONCLUSIONS: Our findings support that bladder PUC is a highly aggressive disease. The lack of E-cadherin expression in PUCs may underlie the distinct discohesive histologic appearance, and abnormal function of the RB gene may be implicated in the development of PUC.

Rrp6: Integrated roles in nuclear RNA metabolism and transcription termination.

The yeast RNA exosome is a eukaryotic ribonuclease complex essential for RNA processing, surveillance, and turnover. It is comprised of a barrel-shaped core and cap as well as a 3'-5' ribonuclease known as Dis3 that contains both endo- and exonuclease domains. A second exonuclease, Rrp6, is added in the nucleus. Dis3 and Rrp6 have both shared and distinct roles in RNA metabolism, and this review will focus primarily on Rrp6 and the roles of the RNA exosome in the nucleus. The functions of the nuclear exosome are modulated by cofactors and interacting partners specific to each type of substrate. Generally, the cofactor TRAMP (Trf4/5-Air2/1-Mtr4 polyadenylation) complex helps unwind unstable RNAs, RNAs requiring processing such as rRNAs, tRNAs, or snRNAs or improperly processed RNAs and direct it toward the exosome. In yeast, Rrp6 interacts with Nrd1, the cap-binding complex, and RNA polymerase II to aid in nascent RNA processing, termination, and polyA tail length regulation. Recent studies have shown that proper termination and processing of short, noncoding RNAs by Rrp6 is particularly important for transcription regulation across the genome and has important implications for regulation of diverse processes at the cellular level. Loss of proper Rrp6 and exosome activity may contribute to various pathologies such as autoimmune disease, neurological disorders, and cancer. WIREs RNA 2016, 7:91-104. doi: 10.1002/wrna.1317 For further resources related to this article, please visit the WIREs website.

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes.

Analysis and significance of c-MET expression in adenoid cystic carcinoma of the salivary gland.

Adenoid cystic carcinoma (ACC), a rare salivary gland malignancy, is a histogenetic, morphologic, and clinical heterogeneous disease. Extensive efforts have been made to characterize molecular events associated with these tumors, including the identification of prognostic and predictive biomarkers. Increased copy number gain and amplification of c-Met, the cell surface receptor for hepatocyte growth factor, has been shown to enhance tumor growth and invasiveness and promote metastasis in certain tumor types. In this study, we evaluated the expression of c-Met by immunohistochemistry (IHC) in a large cohort of salivary gland ACCs and examined its clinicopathologic implications. Archival formalin-fixed paraffin-embedded blocks from 200 ACC patients were used in this study. Pathologic patterns and phenotypic expression of c-Met were recorded and compared with clinical factors including gender, age, disease stage at diagnosis, and clinical outcomes. Correlations between c-MET expression and clinical characteristics were assessed by Pearson's chi-square test or by the 2-tailed Fisher exact test. Curves describing overall survival were generated by Kaplan-Meier product limit method. Strong c-MET expression was seen in inner ductal and outer myoepithelial cells in 53.2% of the cases. There was no correlation between c-Met overexpression and clinicopathologic parameters or patient's overall survival ( p = .94074). In conclusion, c-MET expression is high in a significant subgroup of ACC patients. While c-MET expression is not a prognostic factor in ACC, its role as a predictive marker of benefit from MET inhibitors deserves further investigation.

Child death in the United States: productivity and the economic burden of parental grief.

This article examines the economic consequences associated with the death of a child. The economic costs (funeral and medical expenses and productivity losses) of child death 6 months following the death were estimated based on 213 parents who had experienced the death of a child (usually unexpectedly and predominantly mothers). Findings suggest that productivity losses associated with child death comprise most of the costs and that the economic effects are substantial. Costs associated with on-the-job productivity losses ("presenteeism") outweigh the costs associated with absenteeism. To date, no research has empirically measured both absenteeism and presenteeism following bereavement.

The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae.

The phosphatase Rtr1 has been implicated in dephosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) during transcription elongation and in regulation of nuclear import of RNAPII. Although it has been shown that Rtr1 interacts with RNAPII in yeast and humans, the specific mechanisms that underlie Rtr1 recruitment to RNAPII have not been elucidated. To address this, we have performed an in-depth proteomic analysis of Rtr1 interacting proteins in yeast. Our studies revealed that hyperphosphorylated RNAPII is the primary interacting partner for Rtr1. To extend these findings, we performed quantitative proteomic analyses of Rtr1 interactions in yeast strains deleted for CTK1, the gene encoding the catalytic subunit of the CTD kinase I (CTDK-I) complex. Interestingly, we found that the interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains. We hypothesize that serine-2 CTD phosphorylation is required for Rtr1 recruitment to RNAPII during transcription elongation.