Parents' Experiences with Postpartum Support Groups using Videoconferencing: Perceptions of Safety in the Virtual Space.

PURPOSE: To explore new parents' experiences with web-based videoconferencing as a mechanism of offering postpartum virtual support groups. STUDY DESIGN AND METHODS: Virtual support sessions and individual interviews were conducted to explore participants' experiences with virtual postpartum groups. RESULTS: Thirty-seven parents participated in seven virtual support sessions and 19 participated in individual interviews. Participant narratives centered on perceptions of safety when engaging in virtual support groups. Tools within the virtual space (camera; mute) created a relational paradox which provided safeguards but also hindered the building of trust. Participants described negotiating the fear of harm and judgment within virtual spaces alongside feelings of security in connecting from the safety of their homes. CLINICAL IMPLICATIONS: The virtual environment provides a forum for new parents to access information and support and an avenue for engagement with maternal child nurses and care providers. Awareness of how parents perceive safety in the virtual environment is an important part of facilitating and structuring parent groups on videoconferencing platforms. Nurses should be familiar with videoconferencing technology and be able to guide parents. Experience facilitating virtual groups to ensure safety and security while providing needed support is a valuable nursing skill.

Examining How Postpartum Videoconferencing Support Sessions Can Facilitate Connections between Parents: A Poststructural and Sociomaterial Analysis.

Postpartum support for new parents can normalize experiences, increase confidence, and lead to positive health outcomes. While in-person gatherings may be the preferred choice, not all parents can or want to join parenting groups in person. Online asynchronous chat spaces for parents have increased over the past 10 years, especially during the COVID pandemic, when "online" became the norm. However, synchronous postpartum support groups have not been as accessible. The purpose of our study was to examine how parents experienced postpartum videoconferencing support sessions. Seven one-hour videoconferencing sessions were conducted with 4-8 parents in each group (n = 37). Nineteen parents from these groups then participated in semi-structured interviews. Feminist poststructuralism and sociomaterialism were used to guide the research process and analysis. Parents used their agency to actively think about and interact using visual (camera) and audio (microphone) technologies to navigate socially constructed online discourses. Although videoconferencing fostered supportive connections and parents felt less alone and more confident, the participants also expressed a lack of opportunities for individual conversations. Nurses should be aware of the emerging opportunities that connecting online may present. This study was not registered.

"There's an Etiquette to Zoom That's Not Really Present In-Person": A Qualitative Study Showing How the Mute Button Shapes Virtual Postpartum Support for New Parents.

Virtual spaces that allow parents in the postpartum period to connect, support each other, and exchange information have been increasing in popularity. With the COVID-19 pandemic, many parents had to rely on virtual platforms as a primary means to connect with others and attend to their postpartum health. This study explored virtual postpartum support sessions through the web-based videoconferencing software, Zoom. Guided by feminist poststructuralism and sociomaterialism, we held seven virtual support sessions for parents caring for a baby 0-12 months in age, in Canada, and interviewed 19 participants about their experiences in the sessions. Our methodological approach allowed us to analyze discourses of (1) parenthood, (2) material realities of virtual environments, and (3) support and information on this virtual platform. The purpose of this research was to understand how technology influences postpartum support and learning through online videoconferencing for parents. Our findings document an overarching discourse of Zoom etiquette by which muting was a discursive practice that all participants used. The consistent use of the mute button while not talking structured conversation in virtual postpartum sessions and resulted in three themes: (1) minimizing disruptions; (2) taking turns; and (3) staying on task. The norm of using the mute button changed how parents received and gave support and information. Based on findings and broader literature, we discuss considerations for facilitation of virtual postpartum support sessions.

PERM1 regulates energy metabolism in the heart via ERRα/PGC-1α axis.

Aims: PERM1 is a striated muscle-specific regulator of mitochondrial bioenergetics. We previously demonstrated that PERM1 is downregulated in the failing heart and that PERM1 positively regulates metabolic genes known as targets of the transcription factor ERRα and its coactivator PGC-1α in cultured cardiomyocytes. The aims of this study were to determine the effect of loss of PERM1 on cardiac function and energetics using newly generated Perm1-knockout (Perm1 -/-) mice and to investigate the molecular mechanisms of its transcriptional control. Methods and results: Echocardiography showed that ejection fraction and fractional shortening were lower in Perm1 -/- mice than in wild-type mice (both p < 0.05), and the phosphocreatine-to-ATP ratio was decreased in Perm1 -/- hearts (p < 0.05), indicating reduced contractile function and energy reserves of the heart. Integrated proteomic and metabolomic analyses revealed downregulation of oxidative phosphorylation and upregulation of glycolysis and polyol pathways in Perm1 -/- hearts. To examine whether PERM1 regulates energy metabolism through ERRα, we performed co-immunoprecipitation assays, which showed that PERM1 bound to ERRα in cardiomyocytes and the mouse heart. DNA binding and reporter gene assays showed that PERM1 was localized to and activated the ERR target promoters partially through ERRα. Mass spectrometry-based screening in cardiomyocytes identified BAG6 and KANK2 as potential PERM1's binding partners in transcriptional regulation. Mammalian one-hybrid assay, in which PERM1 was fused to Gal4 DNA binding domain, showed that the recruitment of PERM1 to a gene promoter was sufficient to activate transcription, which was blunted by silencing of either PGC-1α, BAG6, or KANK2. Conclusion: This study demonstrates that PERM1 is an essential regulator of cardiac energetics and function and that PERM1 is a novel transcriptional coactivator in the ERRα/PGC-1α axis that functionally interacts with BAG6 and KANK2.

Sutureless circumcision using monopolar diathermy and 2-octyl cyanoacrylate is safe and effective in a cohort of 634 post-neonatal prepubescent boys.

BACKGROUND: Circumcision is the most commonly performed surgical procedure in the world, with one-third of males circumcised globally. Post-neonatal, prepubescent sutured circumcision demonstrates complication rates ranging from 1.7% to 9.1%. We have previously reported that 2-octyl cyanoacrylate (2-OCA, Dermabond, Ethicon) primary circumcision (PC) and circumcision revision (CR) in prepubescent children demonstrated superior cosmesis, shorter operating room (OR) times and cost savings. OBJECTIVE: The aim of our study is to evaluate complication and reoperation rates with a scalpel-free and suture-less technique for primary circumcision (PC) and circumcision revision (CR) using 2-OCA. METHODS: Following IRB approval, we conducted a retrospective review of all boys at our institution who underwent PC or CR using 2-OCA and monopolar diathermy between January 2014 and January 2021. All procedures were performed by a single surgeon. The technique is outlined in the figure below. No aligning sutures or instruments were used in this process. Patients that required sutures or compressive dressings based on age or associated anomalies were excluded from analysis. We obtained all returns to our system within 30 days of the procedure and returns to the OR during the study period using the REDCap database. RESULTS: Of 1107 procedures performed during the study period, 634 procedures (479 PC and 155 CR) met inclusion criteria. Median age was 12 months (range 3 months-10.4 years) with minimum follow up of 1 year. There were 3 patients (0.47%) that returned to system within 30 days for surgical site bleeding, and one patient (0.15%) required surgical intervention within 30 days. Nine patients required reoperation after 30 days, five (0.8%) for iatrogenic phimosis, 3 (0.5%) for redundant prepuce and 1 for keloid formation. We observed an overall complication rate of 1.3% (6/634) and reoperation rate of 1.6% (10/634). DISCUSSION: Since FDA approval in 1998, 2-OCA has been widely adopted as a replacement for sutures in tension-free wounds. However, it has yet to gain widespread use for circumcision given concerns for wound dehiscence and surgical site bleeding. This study demonstrates that a scalpel-free and suture-less technique demonstrates complication and reoperation rates are lower than reported circumcision using scalpel and sutures. Limitations include retrospective design, single surgeon experience, and REDCAP database only identifying patients with complications that required a return to system. CONCLUSION: Our technique for suture-less circumcision using 2-OCA offers superior cosmesis, shorter OR times, cost savings, and a lower complication rate (1.3%) compared to sutured circumcision (>1.7%) reported in post-neonatal prepubescent boys.

Barriers to and strategies to address COVID-19 testing hesitancy: a rapid scoping review.

BACKGROUND: Testing is a foundational component of any COVID-19 management strategy; however, emerging evidence suggests that barriers and hesitancy to COVID-19 testing may affect uptake or participation and often these are multiple and intersecting factors that may vary across population groups. To this end, Health Canada's COVID-19 Testing and Screening Expert Advisory Panel commissioned this rapid review in January 2021 to explore the available evidence in this area. The aim of this rapid review was to identify barriers to COVID-19 testing and strategies used to mitigate these barriers. METHODS: Searches (completed January 8, 2021) were conducted in MEDLINE, Scopus, medRxiv/bioRxiv, Cochrane and online grey literature sources to identify publications that described barriers and strategies related to COVID-19 testing. RESULTS: From 1294 academic and 97 grey literature search results, 31 academic and 31 grey literature sources were included. Data were extracted from the relevant papers. The most cited barriers were cost of testing; low health literacy; low trust in the healthcare system; availability and accessibility of testing sites; and stigma and consequences of testing positive. Strategies to mitigate barriers to COVID-19 testing included: free testing; promoting awareness of importance to testing; presenting various testing options and types of testing centres (i.e., drive-thru, walk-up, home testing); providing transportation to testing centres; and offering support for self-isolation (e.g., salary support or housing). CONCLUSION: Various barriers to COVID-19 testing and strategies for mitigating these barriers were identified. Further research to test the efficacy of these strategies is needed to better support testing for COVID-19 by addressing testing hesitancy as part of the broader COVID-19 public health response.

Mental Health Impacts of Climate Change on Women: a Scoping Review.

Climate change is the largest threat to human health of the twenty-first century. Women are disproportionately affected by climate change. While the physical health impacts of climate change are an active area of research, works related to the mental health impacts are less developed. Furthermore, the mental health impacts of climate change on women are a particular area of interest due to women's disproportionately negative experiences with climate change and climate change-related events. Therefore, the purpose of this scoping review is to understand what is known from the existing literature regarding the mental health impacts of climate change on women. The methods for this review follow the Arksey and O'Malley framework for a scoping review. By searching databases for publications that discuss women, mental health, and climate change, and screening for relevant work, 20 studies that met inclusion criteria were included in the review. Themes derived from the reviewed studies include negative mental health outcomes, gender-based violence, burdens of care and responsibility, attachment to land and traditions, and the importance of intersectionality. From these findings, there is a clear need for climate policies on adaptation and mitigation to reflect women's unique needs to ensure their health and safety.

Phosphoproteomic analysis of cocaine memory extinction and reconsolidation in the nucleus accumbens.

RATIONALE: Environmental stimuli, or cues, associated with the use of drugs such as cocaine are one of the primary drivers of relapse. Thus, identifying mechanisms to reduce the motivational properties of drug cues is an important research goal. OBJECTIVES: The purpose of this study was to identify cellular signaling events in the nucleus accumbens (NAc) that are induced when a cocaine cue memory is either extinguished through repeated cue presentation in the absence of drug, or when the memory is reactivated and reconsolidated by a brief cue re-exposure. Signaling events specific to extinction or reconsolidation represent potential targets for pharmacotherapeutics that may enhance extinction or disrupt reconsolidation to reduce the likelihood of relapse. METHODS: Male Sprague-Dawley rats were trained to self-administer cocaine paired with an audiovisual cue. Following a period of self-administration, the memory for the cocaine-associated cue was either extinguished, reactivated, or not manipulated (control) 15 min before sacrifice. Tissue from the NAc was subsequently analyzed using mass spectrometry based phosphoproteomics to identify cellular signaling events induced by each condition. RESULTS: Extinction and reconsolidation of the cocaine cue memory produced both common and distinct changes in protein phosphorylation. Notably, there were no significant changes in protein phosphorylation that were modulated in the opposite direction by the two behavioral conditions. Comparison of NAc phosphoproteomic changes to previously identified changes in the basolateral amygdala (BLA) revealed that cue extinction increases phosphorylation at serine (S) 883 of the GABAB receptor subunit 2 and on S14 of syntaxin 1a in both regions, while no common regional signaling events were identified in the reconsolidation group. CONCLUSIONS: Phosphoproteomics is a useful tool for identifying signaling cascades involved in different memory processes and revealed novel potential targets for selectively targeting extinction versus reconsolidation of a cocaine cue memory. Furthermore, cross region analysis suggests that cue extinction may produce unique signaling events associated with increased inhibitory signaling.

Phosphoproteomic Analysis of the Amygdala Response to Adolescent Glucocorticoid Exposure Reveals G-Protein Coupled Receptor Kinase 2 as a Target for Reducing Motivation for Alcohol.

Early life stress is associated with risk for developing alcohol use disorders (AUDs) in adulthood. Though the neurobiological mechanisms underlying this vulnerability are not well understood, evidence suggests that aberrant glucocorticoid and noradrenergic system functioning play a role. The present study investigated the long-term consequences of chronic exposure to elevated glucocorticoids during adolescence on the risk of increased alcohol-motivated behavior, and on amygdalar function in adulthood. A discovery-based analysis of the amygdalar phosphoproteome using mass spectrometry was employed, to identify changes in function. Adolescent corticosterone (CORT) exposure increased alcohol, but not sucrose, self-administration, and enhanced stress-induced reinstatement with yohimbine in adulthood. Phosphoproteomic analysis indicated that the amygdala phosphoproteome was significantly altered by adolescent CORT exposure, generating a list of potential novel mechanisms involved in the risk of alcohol drinking. In particular, increased phosphorylation at serines 296⁻299 on the α2A adrenergic receptor (α2AAR), mediated by the G-protein coupled receptor kinase 2 (GRK2), was evident after adolescent CORT exposure. We found that intra-amygdala infusion of a peptidergic GRK2 inhibitor reduced alcohol seeking, as measured by progressive ratio and stress reinstatement tests, and induced by the α2AAR antagonist yohimbine. These results suggest that GRK2 represents a novel target for treating stress-induced motivation for alcohol which may counteract alterations in brain function induced by adolescent stress exposure.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Quantification of Urinary Protein Biomarkers of Autosomal Dominant Polycystic Kidney Disease by Parallel Reaction Monitoring.

PURPOSE: Autosomal dominant polycystic kidney disease (ADPKD) is a life-long disease in which the genes responsible are known, but the pathogenesis of cyst formation and cyst growth are not understood. Cyst growth ultimately leads to end-stage renal failure in most patients. Analysis of the urinary proteome offers the potential to identify proteins that indicate the presence of cysts (and thus provides diagnosis) as well as the rates of cyst growth (providing prognostic information). EXPERIMENTAL DESIGN: A scheduled parallel reaction monitoring (sPRM) assay is performed on urine samples from 14 patients and 18 normal controls. For relative quantification, stable isotope-labeled synthetic peptides are spiked in the urinary protein digests prior to data collection. The data are subsequently normalized to creatinine and protein concentration in the respective urine samples to control for variations in water intake between individuals. RESULTS: Out of the 143 urinary proteins targeted for sPRM assay, 69 proteins are observed to be significantly dysregulated in ADPKD. The dysregulated proteins are used to cluster ADPKD patients into those who are more or less similar to normal controls. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that sPRM is a promising approach to rapidly screen large numbers of proteins in urine in order to provide earlier diagnosis and potentially better understand the pathogenesis of ADPKD development and progression.

Evaluation of the Nicotinic Acetylcholine Receptor-Associated Proteome at Baseline and Following Nicotine Exposure in Human and Mouse Cortex.

Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein-protein interactions of high-affinity nAChRs containing the ß2 subunit (ß2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the ß2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.

Phosphoproteomic Analysis Reveals a Novel Mechanism of CaMKIIα Regulation Inversely Induced by Cocaine Memory Extinction versus Reconsolidation.

UNLABELLED: Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT: Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance current strategies for addiction treatment.

Development of a Targeted Urine Proteome Assay for kidney diseases.

PURPOSE: Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases. EXPERIMENTAL DESIGN: Potential biomarkers were identified by using a multiproteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope labeled internal peptide standards were synthesized for scheduled MRM. RESULTS: The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least two transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins. CONCLUSIONS AND CLINICAL RELEVANCE: TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from among 167 urinary proteins. This approach, while developed using differentially expressed urinary proteins from patients with delayed versus immediate graft function after kidney transplant, can be expanded to include differentially expressed urinary proteins in multiple kidney diseases. Thus, TUPA could provide a single assay to help diagnose, prognose, and manage many kidney diseases.

MAP4K4 Is a Threonine Kinase That Phosphorylates FARP1.

Mitogen-activated protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with in vitro assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP motifs were predominant among them. In contrast, in vitro assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1.

YPED: an integrated bioinformatics suite and database for mass spectrometry-based proteomics research.

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography-tandem mass spectrometry (LC-MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation.

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K(+) reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K(+) secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)--components of an E3 ubiquitin ligase complex that targets WNKs for degradation--cause constitutively increased renal salt reabsorption and impaired K(+) secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3(S433) phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K(+) secretion in the setting of volume depletion.

Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4.

Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syndrome featuring hypertension and hyperkalemia resulting from constitutive renal salt reabsorption and impaired K(+) secretion. Recently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), components of an E3 ubiquitin ligase complex, were found to cause PHAII, suggesting that loss of this complex's ability to target specific substrates for ubiquitination leads to PHAII. By MS and coimmunoprecipitation, we show that KLHL3 normally binds to WNK1 and WNK4, members of WNK (with no lysine) kinase family that have previously been found mutated in PHAII. We show that this binding leads to ubiquitination, including polyubiquitination, of at least 15 specific sites in WNK4, resulting in reduced WNK4 levels. Dominant disease-causing mutations in KLHL3 and WNK4 both impair WNK4 binding, ubiquitination, and degradation. WNK4 normally induces clearance of the renal outer medullary K(+) channel (ROMK) from the cell surface. We show that WT but not mutant KLHL3 inhibits WNK4-induced reduction of ROMK level. We show that PHAII-causing mutations in WNK4 lead to a marked increase in WNK4 protein levels in the kidney in vivo. These findings demonstrate that CUL3-RING (really interesting new gene) ligases that contain KLHL3 target ubiquitination of WNK4 and thereby regulate WNK4 levels, which in turn regulate levels of ROMK. These findings reveal a specific role of CUL3 and KLHL3 in electrolyte homeostasis and provide a molecular explanation for the effects of disease-causing mutations in both KLHL3 and WNK4.

Proteomics and the analysis of proteomic data: 2013 overview of current protein-profiling technologies.

Mass spectrometry has become a major tool in the study of proteomes. The analysis of proteolytic peptides and their fragment ions by this technique enables the identification and quantitation of the precursor proteins in a mixture. However, deducing chemical structures and then protein sequences from mass-to-charge ratios is a challenging computational task. Software tools incorporating powerful algorithms and statistical methods improved our ability to process the large quantities of proteomics data. Repositories of spectral data make both data analysis and experimental design more efficient. New approaches in quantitative and statistical proteomics make possible a greater coverage of the proteome, the identification of more post-translational modifications, and a greater sensitivity in the quantitation of targeted proteins.

The Ebola virus VP24 protein prevents hnRNP C1/C2 binding to karyopherin α1 and partially alters its nuclear import.

The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon (IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibits phospho-STAT1 nuclear import. A biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) nuclear import as an additional target of VP24. Co-immunoprecipitation studies demonstrate that hnRNP C1/C2 interacts with multiple KPNA family members, including KPNA1. Interaction with hnRNP C1/C2 occurs through the same KPNA1 C-terminal region (amino acids 424-457) that binds VP24 and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 is diminished in the presence of VP24, and cells transiently expressing VP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. These data further define the mechanism of hnRNP C1/C2 nuclear import and demonstrate that the impact of EBOV VP24 on nuclear import extends beyond STAT1.