N6-methyladenosine modification is not a general trait of viral RNA genomes.

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m6A in CHIKV and DENV RNAs. For this, we combine m6A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m6A machinery's localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

The Connell Stitch: A Two-Generation Contribution to Gastrointestinal Surgery.

The history of the Connell Stitch begins at the Milwaukee County Hospital in 1887 and continues across two generations of surgeons, Dr. M.E. Connell and Dr. F Gregory Connell. With this historical article, we review the evolution of the Connell stitch in context of the surgeons responsible for the stitch's development and evolution. Understanding the history of the Connell stitch facilitates a better appreciation for the Connell Stitch that we know and use today.

m6A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication.

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection.

Wrist Arthrodesis Using the Medartis Carpometacarpal Joint Sparing Plate.

BACKGROUND: Total wrist arthrodesis is a well-established surgical technique that provides reliable pain relief in patients with advanced wrist disease. Key limitations of existing plating systems include hardware pull-out, hardware failure, and nonunion. There is limited literature on the newer style carpometacarpal joint (CMCJ) sparing plating system, produced by Medartis. The objective of this study was to determine the long-term clinical and radiological outcomes of wrist arthrodesis with a CMCJ sparing wrist plate. METHODS: This study retrospectively identified 23 wrist arthrodeses using the Medartis CMCJ sparing plate for review. This study assessed the outcomes of 18 unilateral wrist fusions and 1 bilateral wrist fusion. The study group consisted of 12 men and 5 women with an average age of 56 years (range: 29-82 years) with a mean follow-up period of 17 months. RESULTS: At the time of follow-up, all patients' wrists had fused without postsurgical complication. The final grip strength in the operative hand was 28.2 kg/cm2, which was 87% of the contralateral side. The mean Quick Disabilities of the Arm, Shoulder, and Hand score at follow-up was 23.9, with all patients returning to daily living activities and work. Patients reported minimal pain (1.3/10), with almost all (17/18) satisfied with the outcome of the surgery and describing that they would recommend this procedure. CONCLUSION: Our case series highlights that the Medartis wrist arthrodesis plate is a newer design that is a well-tolerated option for wrist arthrodesis based on clinical assessment, functional hand assessment, and patient satisfaction, when compared with the existing literature on traditional plating systems.

Pilot Examination of the Efficacy of the Internet-Delivered, Preoperative, Preparation Program (I-PPP).

Limited evidence-based, interactive, Internet-delivered preoperative preparation programs for children and their parents exist. The purpose of this investigation was to compare the Internet-delivered, preoperative program (I-PPP) in alleviating anxiety in children undergoing outpatient surgery delivered alone (I-PPP) and in conjunction with parental presence (I-PPP + parent) to treatment as usual (TAU). 104 children undergoing day surgery procedures at a local hospital and their parents/guardians participated. Primary outcome measures: (a) observer-rated child anxiety and (b) induction compliance. Results demonstrated an interaction between the I-PPP and TAU groups over time, F(1, 64) = 5.11, p = .027, partial η p2 = .07. At anesthetic induction, the I-PPP group demonstrated lower observer-rated anxiety than TAU, F(1, 64) = 4.72, p = .034, η p2 = .07. I-PPP group demonstrated the best anesthesia induction compliance, F(1, 64) = 4.84, p = .031, η p2 = .07. Our findings demonstrate that the I-PPP is an efficacious preoperative preparation intervention for children. The 'real-world' uptake and integration of the I-PPP into pediatric preoperative settings require exploration going forward. Trial retrospectively registered March 2019 (Open Science Registration https://doi.org/10.17605/osf.io/2x8rg ).

The Tudor SND1 protein is an m6A RNA reader essential for replication of Kaposi's sarcoma-associated herpesvirus.

N6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs. m6A is recognised by YTH domain-containing proteins, which selectively bind to m6A-decorated RNAs regulating their turnover and translation. Using an m6A-modified hairpin present in the Kaposi's sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the 'Royal family' as putative m6A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m6A reader. We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the 'Royal family' have m6A-reading ability, greatly increasing their epigenetic functions beyond protein methylation.

Polymer-Ligand-Based ELISA for Robust, High-Throughput, Quantitative Detection of p53 Aggregates.

A growing number of diseases are being linked to protein misfolding and amyloid formation. Recently, p53 was also shown to associate into amyloid aggregates, raising the question of whether cancer development is associated with protein aggregation as well. However, a lack of suitable tools has hampered the evaluation of their clinical relevance. Herein, we report an enzyme-linked-immunosorbent-assay (ELISA) system based on a polyionic, high-molecular-weight ligand that specifically captures aggregated oligomers and amyloid proteins. We proved that naturally occurring tetramers of p53 are not bound, but high-molecular-weight aggregates are bound and subsequently detected. For the first time, this assay allows the quantitative detection of p53 aggregates from cell lysates, which was demonstrated using 22 ovarian-cancer cell lines as well as 7 patient-derived tumor tissues. The levels of p53 aggregates within the missense-mutated tissue samples varied more than 12-fold. This simple, robust method allows studying the abundance and clinical relevance of protein aggregates. This could help our understanding of the role of protein misfolding in cancer or even in predicting therapy responses to aggregation-targeting drugs.

The m6A-methylase complex recruits TREX and regulates mRNA export.

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA. This modification has previously been shown to alter the export kinetics for mRNAs though the molecular details surrounding this phenomenon remain poorly understood. Recruitment of the TREX mRNA export complex to mRNA is driven by transcription, 5' capping and pre-mRNA splicing. Here we identify a fourth mechanism in human cells driving the association of TREX with mRNA involving the m6A methylase complex. We show that the m6A complex recruits TREX to m6A modified mRNAs and this process is essential for their efficient export. TREX also stimulates recruitment of the m6A reader protein YTHDC1 to the mRNA and the m6A complex influences the interaction of TREX with YTHDC1. Together our studies reveal a key role for TREX in the export of m6A modified mRNAs.

Fully automated, multi-criterial planning for Volumetric Modulated Arc Therapy - An international multi-center validation for prostate cancer.

BACKGROUND AND PURPOSE: Reported plan quality improvements with autoplanning of radiotherapy of the prostate and seminal vesicles are poor. A system for automated multi-criterial planning has been validated for this treatment in a large international multi-center study. The system is configured with training plans using a mechanism that strives for quality improvements relative to those plans. MATERIAL AND METHODS: Each of the four participating centers included thirty manually generated clinical Volumetric Modulated Arc Therapy prostate plans (manVMAT). Ten plans were used for autoplanning training. The other twenty were compared with an automatically generated plan (autoVMAT). Plan evaluations considered dosimetric plan parameters and blinded side-by-side plan comparisons by clinicians. RESULTS: With equivalent Planning Target Volume (PTV) V95%, D2%, D98%, and dose homogeneity autoVMAT was overall superior for rectum with median differences of 3.4 Gy (p < 0.001) in Dmean, 4.0% (p < 0.001) in V60Gy, and 1.5% (p = 0.001) in V75Gy, and for bladder Dmean (0.9 Gy, p < 0.001). Also the clinicians' plan comparisons pointed at an overall preference for autoVMAT. Advantages of autoVMAT were highly treatment center- and patient-specific with overall ranges for differences in rectum Dmean and V60Gy of [-4,12] Gy and [-2,15]%, respectively. CONCLUSION: Observed advantages of autoplanning were clinically relevant and larger than reported in the literature. The latter is likely related to the multi-criterial nature of the applied autoplanning algorithm, with for each center a dedicated configuration that aims at plan improvements relative to its (clinical) training plans. Large variations among patients in differences between manVMAT and autoVMAT point at inconsistencies in manual planning.

Molecular profiling and combinatorial activity of CCT068127: a potent CDK2 and CDK9 inhibitor.

Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer.

Using parathyroid hormone spikes during parathyroidectomy to guide intraoperative decision-making.

BACKGROUND: Intraoperative parathyroid hormone (IOPTH) level monitoring is a useful adjunct to parathyroidectomy for primary hyperparathyroidism (pHPT). Occasionally, increases ("spikes") in IOPTH levels from the preoperative baseline parathyroid hormone may occur, which may lead to longer operative times or more extensive neck exploration or both. The aim of this study was to determine if the extent of IOPTH level increase predicts single gland disease (SGD). METHODS: This is a retrospective review of a prospective parathyroid database of patients undergoing parathyroidectomy for sporadic pHPT from 1999-2013. Extent of parathyroid hormone spike was calculated by the difference in IOPTH level at the time of gland excision and baseline: group 1 had a decrease in IOPTH level, group 2 had IOPTH level increase one to three times above the baseline, and group 3 had IOPTH level increase greater than three times above the baseline. RESULTS: Of the 900 patients in the cohort, there were 634 patients (70%) in group 1, 234 (26%) in group 2, and 32 (4%) in group 3. SGD was identified in 88%, 78%, and 100% of patients in groups 1, 2, and 3, respectively. The median gland weight in group 3 (920 mg) was significantly larger than those in groups 1 and 2 (440 and 460 mg, respectively; P < 0.001). CONCLUSIONS: IOPTH level spikes occur in nearly one-third of patients undergoing parathyroidectomy for sporadic pHPT. Patients with extensive IOPTH level increase are more likely to have larger SGD, whereas patients with moderate IOPTH level increases have increased incidence of multigland disease. In patients with a significant increase in IOPTH levels and larger glands, no further surgical exploration may be indicated.

The role of TREX in gene expression and disease.

TRanscription and EXport (TREX) is a conserved multisubunit complex essential for embryogenesis, organogenesis and cellular differentiation throughout life. By linking transcription, mRNA processing and export together, it exerts a physiologically vital role in the gene expression pathway. In addition, this complex prevents DNA damage and regulates the cell cycle by ensuring optimal gene expression. As the extent of TREX activity in viral infections, amyotrophic lateral sclerosis and cancer emerges, the need for a greater understanding of TREX function becomes evident. A complete elucidation of the composition, function and interactions of the complex will provide the framework for understanding the molecular basis for a variety of diseases. This review details the known composition of TREX, how it is regulated and its cellular functions with an emphasis on mammalian systems.

Analysis of an institutional protocol for thyroid lobectomy: Utility of routine intraoperative frozen section and expedited (overnight) pathology.

BACKGROUND: Intraoperative frozen section (FS) often is performed in patients who undergo thyroid lobectomy to determine the need for completion thyroidectomy. At our institution, if FS pathology is benign, final pathology is expedited overnight. The aim of this study was to determine the utility of FS and to identify a cost-effective management algorithm for thyroid lobectomy. METHODS: A retrospective review was performed of patients who underwent thyroid lobectomy between January 2009 and May 2013. Preoperative cytology ranged from "benign" to "suspicious for malignancy." Clinically significant cancers were defined as >1 cm in size, or multifocal microcarcinomas. RESULTS: Of the 192 patients who underwent thyroid lobectomy with FS, FS was suspicious for malignancy in 5 (3%) patients; 1 (0.5%) underwent immediate completion thyroidectomy. On final pathology, 9 (5%) patients had clinically significant cancers and underwent completion thyroidectomy. FS had a sensitivity and positive predictive value of 22% and 40%, respectively, in identifying clinically significant thyroid cancer. Cost of thyroid lobectomy at varying rates of same-day discharge favored thyroid lobectomy without FS but with expedited pathology for all scenarios. CONCLUSION: At our institution, there appears to be limited utility of FS at the time of thyroid lobectomy given the low predictive value for diagnosing a clinically significant thyroid cancer. In patients who are admitted overnight, expedited pathology is slightly less costly and may improve patient quality-of-life and decrease costs by avoiding delayed completion thyroidectomy. Overnight pathology for patients who undergo thyroid lobectomy may achieve modest cost-savings depending on institutional FS results and rates of malignancy.

Specific loss of CatSper function is sufficient to compromise fertilizing capacity of human spermatozoa.

STUDY QUESTION: Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success? SUMMARY ANSWER: Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF. WHAT IS KNOWN ALREADY: In human spermatozoa, Ca(2+) influx induced by progesterone is mediated by CatSper, a sperm-specific Ca(2+) channel. A suboptimal Ca(2+) influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca(2+)]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception. STUDY DESIGN, SIZE, DURATION: Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were primarily screened using the Ca(2+) influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca(2+) response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca(2+) response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca(2+) response. The mean (± SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca(2+) response. Three men were initially identified with a defective Ca(2+) influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF. LIMITATIONS, REASONS FOR CAUTION: A key limitation relates to working with a specific functional parameter (Ca(2+) influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼ 22% of IVF patients) could not be screened. As such the incidence of significant Ca(2+) abnormalities induced by progesterone may be higher than the ∼ 1% observed here. Additionally, we used a strict definition of a defective Ca(2+) influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca(2+) influx. WIDER IMPLICATIONS OF THE FINDINGS: These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception.

Luzp4 defines a new mRNA export pathway in cancer cells.

Cancer testis antigens (CTAs) represented a poorly characterized group of proteins whose expression is normally restricted to testis but are frequently up-regulated in cancer cells. Here we show that one CTA, Luzp4, is an mRNA export adaptor. It associates with the TREX mRNA export complex subunit Uap56 and harbours a Uap56 binding motif, conserved in other mRNA export adaptors. Luzp4 binds the principal mRNA export receptor Nxf1, enhances its RNA binding activity and complements Alyref knockdown in vivo. Whilst Luzp4 is up-regulated in a range of tumours, it appears preferentially expressed in melanoma cells where it is required for growth.

Low 24-hour urine calcium levels in patients with sporadic primary hyperparathyroidism: is further evaluation warranted prior to parathyroidectomy?

BACKGROUND: Low 24-hour urine calcium (uCa) levels in patients with primary hyperparathyroidism (pHPT) raise concern for familial hypocalciuric hypercalcemia. This study evaluated patients with a low 24-hour uCa level for potential differences that may guide the extent of preoperative evaluation needed. METHODS: A retrospective review was conducted of 1,139 sporadic pHPT patients who underwent parathyroidectomy between December 1999 and May 2011. RESULTS: Of the 54 (5%) patients with greater than or equal to one low 24-hour uCa (<100 mg), 28 (52%) patients had only one low level, 9 (17%) had multiple low levels, and 17 (31%) had a repeat 24-hour uCa greater than 100. In the latter group, 4 of the 9 (53%) patients were on a thiazide and had normalization after cessation. Among the groups, differences existed only in serum creatinine (P = .0011) and glomerular filtration rate (P = .0007). CONCLUSION: This study suggests that sporadic pHPT patients with low 24-hour uCa levels may not require further evaluation with genetic testing for familial hypocalciuric hypercalcemia, especially if previous eucalcemia is documented.

Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions.

GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72-) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (χ(2) P = 0.75) or poly-GA dipeptide repeat protein inclusions (χ(2) P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i) functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat protein.

The phosphorylation of endogenous Nedd4-2 In Na(+)-absorbing human airway epithelial cells.

Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalisation / degradation of epithelial Na(+) channel subunits (α-, ß- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser(221), -Ser(327) and -Thr(246). This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na(+) absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na(+) transport. Effects on Nedd4-2 phosphorylation/abundance and the surface expression of ENaC were monitored by western analysis, whilst Na(+) absorption was quantified electrometrically. Acutely (20min) activating PKA in glucocorticoid-deprived (24h) cells increased the abundance of Ser(221)-phosphorylated, Ser(327)-phosphorylated and total Nedd4-2 without altering the abundance of Thr(246)-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, ß- and γ-ENaC and had only a very small effect upon electrogenic Na(+) absorption. Activating PKA (20min) in glucocorticoid-treated (0.2µM dexamethasone, 24h) cells, on the other hand, increased the abundance of Ser(221)-, Ser(327)- and Thr(246)-phosphorylated and total Nedd4-2; increased the surface abundance of α-, ß- and γ-ENaC and evoked a clear stimulation of Na(+) transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na(+) absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.