Basal activity of PINK1 and PRKN in cell models and rodent brain.

The ubiquitin kinase-ligase pair PINK1-PRKN recognizes and transiently labels damaged mitochondria with ubiquitin phosphorylated at Ser65 (p-S65-Ub) to mediate their selective degradation (mitophagy). Complete loss of PINK1 or PRKN function unequivocally leads to early-onset Parkinson disease, but it is debated whether impairments in mitophagy contribute to disease later in life. While the pathway has been extensively studied in cell culture upon acute and massive mitochondrial stress, basal levels of activation under endogenous conditions and especially in vivo in the brain remain undetermined. Using rodent samples, patient-derived cells, and isogenic neurons, we here identified age-dependent, brain region-, and cell type-specific effects and determined expression levels and extent of basal and maximal activation of PINK1 and PRKN. Our work highlights the importance of defining critical risk and therapeutically relevant levels of PINK1-PRKN signaling which will further improve diagnosis and prognosis and will lead to better stratification of patients for future clinical trials.

Substitution of PINK1 Gly411 modulates substrate receptivity and turnover.

The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development.Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.

Multinational Retrospective Central Pathology Review of Neuroblastoma: Lessons Learned to Establish a Regional Pathology Referral Center in Resource-Limited Settings.

CONTEXT.­: Several countries of the Central America and Caribbean region have been sharing regional neuroblastoma (NB) treatment guidelines. However, there is no standardization in the diagnosis, subclassification, or tumor biology to aid in the risk stratification of these patients. OBJECTIVE.­: To examine the histology and assess the accuracy of the local pathology reports; to evaluate the usefulness of manual MYCN immunohistochemistry (IHC); and to use NB as a model to identify the needs to establish a central pathology review (CPR) program in this region. DESIGN.­: A retrospective CPR of specimens derived from patients with a diagnosis of NB and treated under the regional NB guidelines between 2012 and 2017 was conducted, allowing for a comparison between local diagnoses and the CPR diagnoses. Manual MYCN IHC was performed in the confirmed NB specimens and the results compared with known fluorescence in situ hybridization or automated IHC results, when available. RESULTS.­: The 156 specimens reviewed included 460 blocks and 183 original slides. Neuroblastoma was confirmed in 138 samples (88.5%), but low concordance rates for Shimada classification (n = 39; 25.0%), mitotic-karyorrhectic index (n = 4; 2.5%), and International Neuroblastoma Pathology Classification (n = 18; 11.5%) were noted. Manual MYCN IHC performed on 120 specimens showed conclusive results in 89.2% (28 positive, 23.4%; 79 negative, 65.8%) and questionable results in 10.8% (n = 13). CONCLUSIONS.­: This retrospective CPR highlights the need for a CPR program to serve this region, to ensure correct diagnosis and subclassification of NB, and to provide manual MYCN IHC-with reflexing to fluorescence in situ hybridization, if questionable. This approach can further regional collaboration, enhance test utilization, and ultimately improve patients' outcomes.

Dual-specificity phosphatase 29 is induced during neurogenic skeletal muscle atrophy and attenuates glucocorticoid receptor activity in muscle cell culture.

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Newly named dual-specificity phosphatase 29 (Dusp29) has been identified as a novel neurogenic atrophy-induced gene in skeletal muscle. Quantitative PCR analysis revealed that Dusp29 expression is significantly higher in differentiated myotubes compared with proliferating myoblasts. To determine how Dusp29 is transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dusp29 were cloned, fused to a reporter gene, and found to be highly inducible in response to ectopic expression of the myogenic regulatory factors (MRF), MyoD and myogenin. Furthermore, site-directed mutagenesis of conserved E-box elements within the proximal promoter of Dusp29 rendered a Dusp29 reporter gene unresponsive to MRF overexpression. Dusp29, an atypical Dusp also known as Dupd1/Dusp27, was found to attenuate the ERK1/2 branch of the MAP kinase signaling pathway in muscle cells and inhibit muscle cell differentiation when ectopically expressed in proliferating myoblasts. Interestingly, Dusp29 was also found to destabilize AMPK protein while simultaneously enriching the phosphorylated pool of AMPK in muscle cells. Additionally, Dusp29 overexpression resulted in a significant increase in the glucocorticoid receptor (GR) protein and elevation in GR phosphorylation. Finally, Dusp29 was found to significantly impair the ability of the glucocorticoid receptor to function as a transcriptional activator in muscle cells treated with dexamethasone. Identifying and characterizing the function of Dusp29 in muscle provides novel insights into the molecular and cellular mechanisms for skeletal muscle atrophy.

A prospective observational study of postoperative follow-ups and outcomes at a nonprofit, internationally supported pediatric surgery center in Guatemala.

BACKGROUND: Global surgical access is unequally distributed, with the greatest surgical burden in low- and middle-income countries, where surgical care is often supplemented by nongovernmental organizations. Quality data from organizations providing this care are rarely collected or reported. The Moore Pediatric Surgery Center in Guatemala City, Guatemala, is unique in that it offers a permanently staffed, freestanding pediatric surgical center. Visiting surgical teams supplement the local permanent staff by providing a broad range of pediatric subspecialty surgical and anesthesia care. AIM: The aim of this study was to collect and report the incidence of completed postoperative follow-up visits and outcome measures at this nonprofit, internationally supported surgery center. METHODS: De-identified demographic and postoperative outcome data were collected from each routinely scheduled, one-week pediatric surgical mission trip and incorporated into an electronic data collection system. Emphasis was placed on identification of completed postoperative visits and associated perioperative complications. After 27 months of data collection, results were analyzed to identify and quantify trends in patient follow-ups and postoperative outcomes. RESULTS: Over 27 months, 1639 pediatric surgical procedures were performed and included in data analysis. The percentage of completed postoperative day-1 follow-up visits was 99.1%, and seven complications were identified out of these 1624 cases (postoperative complication rate of 0.4%). The percentage of completed first postoperative visits after discharge was 93.3%, and 67 complications were identified out of these 1530 cases (postoperative complication rate of 4.4%). CONCLUSION: Our data show a high rate of postoperative follow-up visits completed and low perioperative complication rates similar to those of high-income countries. Our data suggest that The Moore Surgery Center model of care offers an alternative to the short-term visiting surgical model by incorporating the local system and allows for improved follow-up, outcomes analysis, and high quality of care.

Correlation Between MYCN Gene Status and MYCN Protein Expression in Neuroblastoma: A Pilot Study To Propose the Use of MYCN Immunohistochemistry in Limited-Resource Areas.

PURPOSE: The most significant adverse risk factor for neuroblastoma (NB) is MYCN gene amplification, which strongly associates with high-risk disease. Fluorescent in situ hybridization (FISH) is considered the best method to evaluate MYCN gene status. However, it requires a laboratory that can perform highly complex testing, specialized personnel, and costly reagents. Herein, we aimed to investigate the feasibility of using immunohistochemistry (IHC) to detect MYCN protein expression in lieu of FISH, a strategy potentially useful in areas with limited resources. METHODS: A pilot cohort of 78 patients with NB, including 34 of Middle Eastern descent (MED) who had a higher prevalence of MYCN gene amplification (44.11%) and 44 of North American descent (NAD), nine (20.45%) of whom had MYCN amplification, was evaluated with IHC for MYCN protein. Correlations of FISH results and protein expression are presented. RESULTS: A positive correlation between MYCN gene amplification and protein expression by IHC was seen in 22 (91.66%) of the 24 MYCN-amplified NB cases-14 (93.33%) of 15 patients of MED and eight (88.88%) of nine patients of NAD. Agreement between negative FISH and negative IHC results was noted in 18 (94.73%) patients of MED and 34 (97.14%) patients of NAD. Two cases had weak protein expression but no gene amplification (MED: n = 1; 5.0%; NAD: n = 1; 2.9%). CONCLUSION: An excellent overall correlation between MYCN gene status by FISH and MYCN protein expression by IHC was confirmed. MYCN IHC in NB with reflexing to FISH in equivocal cases is potentially useful in a limited-resource setting. Evaluation of effectiveness using a larger cohort and optimization to perform MYCN IHC manually is needed.

Ttc39c is upregulated during skeletal muscle atrophy and modulates ERK1/2 MAP kinase and hedgehog signaling.

Tetratricopeptide repeat domain containing 39c (Ttc39c) is expressed in skeletal muscle and is transcriptionally activated in response to neurogenic atrophy in mice. Expression analysis using quantitative polymerase chain reaction and Western blots revealed that Ttc39c is expressed in both proliferating and differentiated muscle cells, peaking during early differentiation and then decreasing as cells progress further through the differentiation process. To further analyze the transcriptional regulation of Ttc39c, promoter fragments of the gene were cloned and fused with the secreted alkaline phosphatase reporter gene. The Ttc39c reporter plasmids were then transfected into cultured mouse muscle cells and found to have transcriptional activity. Furthermore, overexpression of MyoD and myogenin resulted in significant transcriptional repression of the Ttc39c reporter genes. To determine subcellular localization, an expression plasmid with the Ttc39c complementary DNA fused with green fluorescent protein was transfected into muscle cells and analyzed by confocal fluorescent microscopy showing that Tct39c localizes exclusively to the cytoplasm of cultured cells. To assess potential function in muscle, Ttc39c was overexpressed leading to vitiated muscle cell differentiation, impaired ERK1/2 MAP Kinase and Hedgehog signaling, and increased expression of IFT144 and IFT43, which are part of the IFT-A complex involved in retrograde transport in primary cilia. Interestingly, Ttc39c knockdown also resulted in inhibition of muscle cell differentiation and impaired Hedgehog and MAP Kinase signaling but did not affect IFT144 or IFT433 expression. The results of this study demonstrate that muscle cell differentiation is sensitive to abnormal Ttc39c expression and that normal Ttc39c expression appears to be necessary for proper MAP Kinase and Hedgehog signal transduction in developing muscle cells.

Dual-specificity phosphatase 4 is upregulated during skeletal muscle atrophy and modulates extracellular signal-regulated kinase activity.

Skeletal muscle atrophy results from disparate physiological conditions, including denervation, corticosteroid treatment, and aging. The purpose of this study was to describe and characterize the function of dual-specificity phosphatase 4 (Dusp4) in skeletal muscle after it was found to be induced in response to neurogenic atrophy. Quantitative PCR and Western blot analysis revealed that Dusp4 is expressed during myoblast proliferation but rapidly disappears as muscle cells differentiate. The Dusp4 regulatory region was cloned and found to contain a conserved E-box element that negatively regulates Dusp4 reporter gene activity in response to myogenic regulatory factor expression. In addition, the proximal 3'-untranslated region of Dusp4 acts in an inhibitory manner to repress reporter gene activity as muscle cells progress through the differentiation process. To determine potential function, Dusp4 was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize to the nucleus of proliferating myoblasts. Furthermore, Dusp4 overexpression delayed C2C12 muscle cell differentiation and resulted in repression of a MAP kinase signaling pathway reporter gene. Ectopic expression of a Dusp4 dominant negative mutant blocked muscle cell differentiation and attenuated MAP kinase signaling by preferentially targeting the ERK1/2 branch, but not the p38 branch, of the MAP kinase signaling cascade in skeletal muscle cells. The findings presented in this study provide the first description of Dusp4 in skeletal muscle and suggest that Dusp4 may play an important role in the regulation of muscle cell differentiation by regulating MAP kinase signaling.

Improving Immunohistochemistry Capability for Pediatric Cancer Care in the Central American and Caribbean Region: A Report From the AHOPCA Pathology Working Group.

Accessibility to immunohistochemistry (IHC) is invaluable to proper diagnosis and treatment of pediatric patients with malignant neoplasms. Whereas IHC is widely available in anatomic pathology laboratories in high-income countries, access to it in anatomic pathology laboratories of low- and middle-income countries remains a struggle, with many limitations. To advance the quality of the pathology service offered to children with cancer in areas with limited resources, a 5-day pathology training workshop was offered to pathologists and histotechnologists from various countries of the Central American and Caribbean region. An initial assessment of the workshop participants' current laboratory capacities was performed, and a regional training center was selected. Didactic and hands-on activities were offered, and review and evaluation of the IHC slides produced during the training course were compared with original slides from the participants' sites. This model of intensive 5-day training appears to be effective and can potentially be used in other budget-constrained regions. Moreover, it can serve as a continuing education activity for pathologists and histotechnologists, and as part of validations and quality improvement projects to build capacity and develop IHC assay proficiency in low- and middle-income countries.

Factors associated with the use of mosquito bed nets: results from two cross-sectional household surveys in Zambézia Province, Mozambique.

BACKGROUND: Malaria remains a major threat to some 3.2 billion persons globally. Malaria contributes heavily to the overall disease burden in Mozambique and is considered endemic. A cornerstone of Mozambique's vector control strategy has been to strive for universal coverage of insecticide-treated nets (ITN). METHODS: The study is a population-based cross-sectional survey of female heads-of-household in Zambézia Province, Mozambique conducted during August-September, 2010 and April-May, 2014. Analyses accounted for a stratified two-stage cluster sample design. Outcomes of interest included sleeping under a mosquito net during the previous night. Descriptive statistics were calculated for three oversampled districts and for the entire province. Multivariable logistic regression analysis was used to estimate factors associated with both changes over time and increased mosquito bed net usage. RESULTS: Of the 3916 households interviewed in 2010 and 3906 households in 2014, 64.3% were in possession of at least one mosquito bed net. A higher proportion of households in Namacurra (90%) reported possession of a mosquito net, compared to Alto Molócuè (77%) and Morrumbala (34%), respectively in 2014. Of pregnant respondents, 58.6% reported sleeping under a mosquito net the previous night in 2010 compared to 68.4% in 2014. Fifty percent of children 0-59 months slept under a mosquito net the previous night in 2010 compared to 60% in 2014. Factors associated with use of a mosquito net for female head-of-household respondents were higher education, understanding Portuguese, larger household size, having electricity in the household, and larger household monthly income. As travel time to a health facility increased (per 1 h), respondents had 13% lower odds of sleeping under a mosquito net (OR 0.87; 95% CI 0.74-1.01, p = 0.07). Pregnant women in 2014 had a 2.4 times higher odds of sleeping under a bed net if they lived in Namacurra compared to Alto Molócuè (95% CI 0.91-6.32, p = 0.002 for district). Higher maternal education, living in Namacurra, and acquisition of mosquito bed nets were associated with a child 0-59 months reporting sleeping under the net in the previous night in 2014. CONCLUSIONS: Intensified focus on the poorest, least educated, and most distant from health services is needed to improve equity of ITN availability and usage. Additionally, while some districts have already surpassed goals in terms of coverage and utilization of ITN, renewed emphasis should be placed on bringing all geographic regions of the province closer to meeting these targets.

Optimism, stress, life satisfaction, and job burnout in restaurant managers.

Researchers have suggested that dispositional optimism is related to both stress and stress outcomes. However, the nature of this relationship has not fully been explained. The authors contributed to a better understanding of this relationship by evaluating dispositional optimism, stress, overall life satisfaction, and job burnout in a sample of restaurant managers. Results indicated that stress and job burnout were significantly related; however, their relationship was not moderated by dispositional optimism, as would be suggested by the results of past research. The diminished personal accomplishment dimension of job burnout mediated the relationship between optimism and life satisfaction. Also, stress significantly impacted perceptions of diminished personal accomplishment and life satisfaction.