Ig Superfamily Ligand and Receptor Pairs Expressed in Synaptic Partners in Drosophila.

Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity.

C9orf72 Poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function.

Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

eIF5 stimulates the CUG initiation of RAN translation of poly-GA dipeptide repeat protein (DPR) in C9orf72 FTLD/ALS.

Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response, but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during integrated stress response, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS.

Systematic expression profiling of Dpr and DIP genes reveals cell surface codes in Drosophila larval motor and sensory neurons.

In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell-surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. Here, we investigated expression of Dpr and DIP genes, two CSP subfamilies belonging to the immunoglobulin superfamily, in Drosophila larval motor neurons (MNs), muscles, glia and sensory neurons (SNs) using a collection of GAL4 driver lines. We found that Dpr genes are more broadly expressed than DIP genes in MNs and SNs, and each examined neuron expresses a unique combination of Dpr and DIP genes. Interestingly, many Dpr and DIP genes are not robustly expressed, but are found instead in gradient and temporal expression patterns. In addition, the unique expression patterns of Dpr and DIP genes revealed three uncharacterized MNs. This study sets the stage for exploring the functions of Dpr and DIP genes in Drosophila MNs and SNs and provides genetic access to subsets of neurons.

Validation of resting full-cycle ratio and diastolic pressure ratio with [15O]H2O positron emission tomography myocardial perfusion.

Fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) are invasive techniques used to evaluate the hemodynamic significance of coronary artery stenosis. These methods have been validated through perfusion imaging and clinical trials. New invasive pressure ratios that do not require hyperemia have recently emerged, and it is essential to confirm their diagnostic efficacy. The aim of this study was to validate the resting full-cycle ratio (RFR) and the diastolic pressure ratio (dPR), against [15O]H2O positron emission tomography (PET) imaging. A total of 129 symptomatic patients with an intermediate risk of coronary artery disease (CAD) were included. All patients underwent cardiac [15O]H2O PET with quantitative assessment of resting and hyperemic myocardial perfusion. Within a 2 week period, coronary angiography was performed. Intracoronary pressure measurements were obtained in 320 vessels and RFR, dPR, and FFR were computed. PET derived regional hyperemic myocardial blood flow (hMBF) and myocardial perfusion reserve (MPR) served as reference standards. In coronary arteries with stenoses (43%, 136 of 320), the overall diagnostic accuracies of RFR, dPR, and FFR did not differ when PET hyperemic MBF < 2.3 ml min-1 (69.9%, 70.6%, and 77.1%, respectively) and PET MPR < 2.5 (70.6%, 71.3%, and 66.9%, respectively) were considered as the reference for myocardial ischemia. Non-significant differences between the areas under the receiver operating characteristic (ROC) curve were found between the different indices. Furthermore, the integration of FFR with RFR (or dPR) does not enhance the diagnostic information already achieved by FFR in the characterization of ischemia via PET perfusion. In conclusion, the novel non-hyperemic pressure ratios, RFR and dPR, have a diagnostic performance comparable to FFR in assessing regional myocardial ischemia. These findings suggest that RFR and dPR may be considered as an FFR alternative for invasively guiding revascularization treatment in symptomatic patients with CAD.

Group citizenship behaviour in healthcare organization, doctor-patient relationship, work engagement and turnover intention: A moderated mediation model.

The effective functioning of health care organisations depends on the inter-professional collaboration among healthcare professionals from diverse backgrounds, representing different work units, to provide quality services. This study aims to understand how group citizenship behaviour (GCB) that supports other work groups may moderate the relationship between doctor-patient relationship (DPR), Work Engagement (WE) and Turnover Intention (TI). The data for this study were collected through two waves of questionnaire survey at a tertiary public hospital in China. The hypothesised model was tested by Hayes' PROCESS macro. There were significant differences of perceived GCB across different professional units with work units practicing a multidisciplinary working approach and working in the high-stake working environment reported higher levels of GCB. The results show that WE mediates the relationships between DPR and TI and GCB accentuates the positive relationship between DPR and WE. Specifically, the path between DPR and WE was stronger for individuals with high perceived GCB than those with low perceived GCB. The study contributes to the development of Job Demands-Resources model with integrating GCB into the model and enriching the challenge job demand conceptualisation by focussing on DPR in Chinese health care setting. In practice, the hospital administrators should encourage GCB to improve organizational effectiveness and doctors' attitudes.

A Symmetrized Dot Pattern Extraction Method Based on Frobenius and Nuclear Hybrid Norm Penalized Robust Principal Component Analysis and Decomposition and Reconstruction.

Due to their symmetrized dot pattern, rolling bearings are more susceptible to noise than time-frequency characteristics. Therefore, this article proposes a symmetrized dot pattern extraction method based on the Frobenius and nuclear hybrid norm penalized robust principal component analysis (FNHN-RPCA) as well as decomposition and reconstruction. This method focuses on denoising the vibration signal before calculating the symmetric dot pattern. Firstly, the FNHN-RPCA is used to remove the non-correlation between variables to realize the separation of feature information and interference noise. After, the residual interference noise, irrelevant information, and fault features in the separated signal are clearly located in different frequency bands. Then, the ensemble empirical mode decomposition is applied to decompose this information into different intrinsic mode function components, and the improved DPR/KLdiv criterion is used to select components containing fault features for reconstruction. In addition, the symmetrized dot pattern is used to visualize the reconstructed signal. Finally, method validation and comparative analysis are conducted on the CWRU datasets and experimental bench data, respectively. The results show that the improved criteria can accurately complete the screening task, and the proposed method can effectively reduce the impact of strong noise interference on SDPs.

The porphyrin TMPyP4 inhibits elongation during the noncanonical translation of the FTLD/ALS-associated GGGGCC repeat in the C9orf72 gene.

GGGGCC (G4C2) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G4C2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G4C2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G4C2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G4C2 repeat RNA. Urea-resistant interaction between G4C2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G4C2 repeat translation elongation.

C9orf72 hexanucleotide repeat expansion leads to altered neuronal and dendritic spine morphology and synaptic dysfunction.

Frontotemporal lobar degeneration (FTLD) comprises a heterogenous group of progressive neurodegenerative syndromes. To date, no validated biomarkers or effective disease-modifying therapies exist for the different clinical or genetic subtypes of FTLD. The most common genetic cause underlying FTLD and amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion in the C9orf72 gene (C9-HRE). FTLD is accompanied by changes in several neurotransmitter systems, including the glutamatergic, GABAergic, dopaminergic, and serotonergic systems and many clinical symptoms can be explained by disturbances in these systems. Here, we aimed to elucidate the effects of the C9-HRE on synaptic function, molecular composition of synapses, and dendritic spine morphology. We overexpressed the pathological C9-HRE in cultured E18 mouse primary hippocampal neurons and characterized the pathological, morphological, and functional changes by biochemical methods, confocal microscopy, and live cell calcium imaging. The C9-HRE-expressing neurons were confirmed to display the pathological RNA foci and DPR proteins. C9-HRE expression led to significant changes in dendritic spine morphologies, as indicated by decreased number of mushroom-type spines and increased number of stubby and thin spines, as well as diminished neuronal branching. These morphological changes were accompanied by concomitantly enhanced susceptibility of the neurons to glutamate-induced excitotoxicity as well as augmented and prolonged responses to excitatory stimuli by glutamate and depolarizing potassium chloride as compared to control neurons. Mechanistically, the hyperexcitation phenotype in the C9-HRE-expressing neurons was found to be underlain by increased activity of extrasynaptic GluN2B-containing N-methyl-d-aspartate (NMDA) receptors. Our results are in accordance with the idea suggesting that C9-HRE is associated with enhanced excitotoxicity and synaptic dysfunction. Thus, therapeutic interventions targeted to alleviate synaptic disturbances might offer efficient avenues for the treatment of patients with C9-HRE-associated FTLD.

Family of neural wiring receptors in bilaterians defined by phylogenetic, biochemical, and structural evidence.

The evolution of complex nervous systems was accompanied by the expansion of numerous protein families, including cell-adhesion molecules, surface receptors, and their ligands. These proteins mediate axonal guidance, synapse targeting, and other neuronal wiring-related functions. Recently, 32 interacting cell surface proteins belonging to two newly defined families of the Ig superfamily (IgSF) in fruit flies were discovered to label different subsets of neurons in the brain and ventral nerve cord. They have been shown to be involved in synaptic targeting and morphogenesis, retrograde signaling, and neuronal survival. Here, we show that these proteins, Dprs and DIPs, are members of a widely distributed family of two- and three-Ig domain molecules with neuronal wiring functions, which we refer to as Wirins. Beginning from a single ancestral Wirin gene in the last common ancestor of Bilateria, numerous gene duplications produced the heterophilic Dprs and DIPs in protostomes, along with two other subfamilies that diversified independently across protostome phyla. In deuterostomes, the ancestral Wirin evolved into the IgLON subfamily of neuronal receptors. We show that IgLONs interact with each other and that their complexes can be broken by mutations designed using homology models based on Dpr and DIP structures. The nematode orthologs ZIG-8 and RIG-5 also form heterophilic and homophilic complexes, and crystal structures reveal numerous apparently ancestral features shared with Dpr-DIP complexes. The evolutionary, biochemical, and structural relationships we demonstrate here provide insights into neural development and the rise of the metazoan nervous system.

Exploring the doctor-patient relationship as a challenge job demand: application of the job demands-resources model in a Chinese public hospital.

In the increasingly commercialized healthcare environment in China, doctor-patient relationship (DPR) is a job demand for doctors that is linked to various motivational outcomes. Drawing on the Job Demands-Resources (JD-R) model, and the conservation of resources theory, we develop a preliminary conceptual model that links Leader Member Exchange (LMX) as a job resource, and DPR as a challenge job demand, to the levels of work engagement and turnover intentions of doctors working in this healthcare environment. Using two-wave data collected from 381 doctors in a public hospital, we found support for the hypothesized model. Results of a series of SEM analyses revealed that LMX was positively related to DPR and work engagement, while DPR partially mediates the path from LMX to work engagement. In addition, LMX is negatively related to turnover intentions through DPR and subsequently work engagement. Theoretically, this study contributes to the development of the JD-R model by investigating the concept of challenge job demand, and its role in the motivational process, with new evidence from healthcare occupations in China. Practically, this study contributes to the limited number of studies on managing the changing nature of the DPR in China, and in seeking potential solutions based on established organizational constructs.

Assessing prognostic value of early tumor shrinkage and depth of response in first-line therapy for patients with advanced unresectable pancreatic cancer.

BACKGROUND: The prognostic potential of early tumor shrinkage (ETS) and depth of response (DpR) in pancreatic cancer (PC) is unclear. Here, we recruited 90 patients with recurrent and metastatic PC (RMPC) who had received chemotherapy as first-line therapy to assess the prognostic potential of these markers. METHODS: ETS is characterized as a ≥ 20% depletion in the sum-of-the-longest-diameters (SLD) of measurable tumor lesions at 6-12 weeks than the baseline. DpR is the maximum shrinkage (%) from the baseline to nadir. We evaluated corrections in ETS and DpR with survival. RESULTS: Of the 63 patients in which ETS assessment was possible, 21 (33.3%) achieved ETS. We found a significant association between the incidence of ETS and an improved rate of progression-free survival (PFS; 6.5 vs. 2.2 months; p < 0.001) and overall survival (OS; 12.1 vs. 6.0 months; p = 0.014). The median value of DpR was - 23.66%. DpR was also related to improved PFS (9.3 vs. 3.1 months; p < 0.001) and OS (18.2 vs. 7.3 months; p < 0.001). Patients who had distant metastasis, not local recurrence, with ETS showed markedly better outcomes. In a multivariate model, both ETS and DpR were independent predictors of OS in the whole population. CONCLUSIONS: ETS and DpR may predict favorable outcomes for RMPC patients who had received chemotherapy as first-line therapy, independent of the agents used. Further studies on the exploratory analyses of the optimum ETS cut-off value in recurrent PC patients to predict favorable clinical outcomes are required.

Investigation of the differences between the medical personnel's and general population's view on the doctor-patient relationship in China by a cross-sectional survey.

BACKGROUND: Due to economic development and an increase in the aging population, the demand for medical resources is increasing. A good doctor-patient relationship (DPR) can optimize patients' medical experience and improve treatment efficiency. The DPR, however, is currently in crisis in China. To explore ways to improve DPR, this study assessed the views on the status of the DPR, medical services, and the general situation of medical work among medical personnel (MP) and the general population (GP). METHODS: This cross-sectional study, conducted between December 2019 and March 2020, targeted the MP and the GP in Nanjing City, Jiangsu Province, and Zhengzhou City, Henan Province. A total of 154 MP and 329 GP answered a self-administered questionnaire through Questionnaire Star and WeChat apps. Wilcoxon's Sign Rank Test, Chi-square test, and frequency distributions and percentages were used to process the data. RESULTS: Only 11.04% of the MP and 14.89% of the GP believed that the current DPR was harmonious. Moreover, 54.55% of the MP and 71.12% of the GP believed that the medical industry was a service industry. While 14.29% of the MP and 64.44% of the GP thought medical staff earned high salaries, 19.48% of the MP and 47.11% of the GP wanted their children to be in the medical industry. The recognition of the current status of the DPR did not affect the GP's preference for their children's practice (p < 0.05). Most MPs hoped to improve salaries (40.26%), followed by safety (17.53%) and social status (12.99%); only 8.44% of the MP wanted to improve the DPR. CONCLUSION: The MP's and GP's views on the current status of DPR, the importance of medical service attitudes, and the general sense of the medical industry were similar. However, there was a significant difference in the perception of the nature of medical services and the income of the people employed in the medical industry between the two groups. Balancing the expectations of patients in the medical industry and increasing public awareness of the actual situation in the medical industry may be a feasible way to improve the DPR.

Relationship between sire predicted transmitting ability for daughter pregnancy rate and daughter's reproductive performance and milk production in Japanese dairy herds.

Modern genetic improvement in dairy cattle is directed towards improvement of fertility; however, reproduction traits generally exhibit a genetic antagonism with milk yield. Herein, we aimed to clarify the effects of sire predicted transmitting ability (PTA) for daughter pregnancy rate (DPR) on the reproductive performance and milk yield of daughters in Japanese dairy herds. We conducted a retrospective cohort study on four dairy herds in eastern Hokkaido, Japan, using 1,612 records from 1,018 cows with first, second, or third calvings between March 2015 and September 2018. First, we classified sires into three groups based on the tertile value of their DPR estimate: ≤ -2.2 (low), -2.1 to -0.4 (intermediate), and ≥ -0.3 (high). Subsequently, we compared the sire PTA estimates, reproductive performance, and milk production among DPR groups for each parity of the daughters. In the first and second parity, the hazard of pregnancy by 200 days postpartum was highest in cows from the high-DPR group (P < 0.05); in the third parity, it was unaffected by DPR group. Although sire PTA for milk production in cows from the low-DPR group was highest, actual milk production was unaffected by DPR group regardless of parity. Our findings demonstrate that using sires with PTA for high fertility can enable farmers to improve reproductive performance without decreasing milk yield in Japanese dairy herds. However, it should be noted that sires with PTA for high fertility are at risk for reducing the genetic merit for milk production.

A Review of the Chemistry, Pesticide Use, and Environmental Fate of Sulfur Dioxide, as Used in California.

Sulfur dioxide (SO2) is an atmospheric pollutant that is moderately persistent in the atmosphere and highly water soluble. When applied as a pesticide, SO2 may be transported, deposited, or transformed in various chemical reactions. SO2 participates in the sulfur biogeochemical cycle, which involves complex reactions of sulfur-containing compounds between abiotic and biotic components of ecosystems. The main degradation route of SO2 is atmospheric oxidation, and sulfur oxides may undergo long-distance transport prior to removal from the atmosphere by wet or dry deposition. According to the Pesticide Use Reporting (PUR) database maintained by the California Department of Pesticide Regulation (DPR), SO2 use in California from 2010 to 2015 was primarily for fumigations (96%), including treatments of postharvest grape products and winery equipment sterilizations. Other site uses contributed less than 5% of reported statewide SO2 use from 2010 to 2015. A slight increasing trend in use of SO2 as a pesticide was observed from 2010 to 2015, with the highest reported uses of SO2 within California counties during the months of July-November. Although the primary sources of SO2 in the environment are anthropogenic emissions from the combustion of fossil fuels, emissions of SO2 from pesticide uses have the potential to contribute to the environmental and public welfare impacts of SO2 pollution. Oxidation of atmospheric SO2 may contribute to the negative environmental and public welfare impacts of acid rain, which include toxicity to aquatic organisms, fish, and terrestrial vegetation, and corrosion of man-made materials.

A computer aided diagnosis system for measurement of mandibular cortical thickness on dental panoramic radiographs in prediction of women with low bone mineral density.

Osteoporosis detection at earlier stages can enhance the life span of an elderly individual. The aim of the study is to perform semi-automated measurement of mandibular cortical thickness (MCT) on a dental panoramic radiograph (DPR) and thereby to predict the risk of low BMD among the studied population. The study involved 76 women (mean age: 57.2 ± 12.6 years). The DPR was obtained using KODAK 8000C system. The BMD of right total hip (T-BMD) was obtained using DPX Prodigy Dual-energy X-ray absorptiometry (DXA) Scanner. The DPR obtained were subjected to image processing techniques to perform MCT measurement. The region of interest was manually selected around the mental foramen and enhanced using a median filter. The Ostu segmentation was performed and connected component labelling operation was performed to determine the lower boundary by finding the contour with maximum area. Subsequently, the haar wavelet operation was carried out to find the magnitude and thereby select the upper delineating cortical boundary. The Pearson test results revealed (r = 0.96, p < 0.01) for the standard (manual) MCT measurement against the MCT measured using the proposed semi-automated scheme. ROC analysis revealed that MCT = 2.5 mm could be an optimal threshold in spotting individuals at risk of low BMD. The results of the study revealed that the MCT measured on a DPR using the proposed approach could be helpful for identifying individuals at risk of low BMD.

Disruption of a Novel Iron Transport System Reverses Oxidative Stress Phenotypes of a dpr Mutant Strain of Streptococcus mutans.

The Dps-like peroxide resistance protein (Dpr) is essential for H2O2 stress tolerance and aerobic growth of the oral pathogen Streptococcus mutans Dpr accumulates during oxidative stress, protecting the cell by sequestering iron ions and thereby preventing the generation of toxic hydroxyl radicals that result from the interaction of iron with H2O2 Previously, we reported that the SpxA1 and SpxA2 regulators positively regulate expression of dpr in S. mutans Using an antibody raised against S. mutans Dpr, we confirmed at the protein level the central and cooperative nature of SpxA1 and SpxA2 regulation in Dpr production. During phenotypic characterization of the S. mutans Δdpr strain, we observed the appearance of distinct colony variants, which sometimes lost the oxidative stress sensitivity typical of Δdpr strains. Whole-genome sequencing of these phenotypically distinct Δdpr isolates revealed that a putative iron transporter operon, smu995-smu998, was a genomic hot spot with multiple single nucleotide polymorphisms identified within the different isolates. Deletion of smu995 or the entire smu995-smu998 operon in the Δdpr background strain completely reversed the oxidative stress-sensitive phenotypes associated with dpr inactivation. Conversely, inactivation of genes encoding the ferrous iron transport system FeoABC did not alleviate phenotypes of the Δdpr strain. Preliminary characterization of strains lacking smu995-smu998, feoABC, and the iron/manganese transporter gene sloABC revealed the interactive nature of these three systems in iron transport but also indicated that there may be additional iron uptake systems in S. mutansIMPORTANCE The dental caries-associated pathogen Streptococcus mutans routinely encounters oxidative stress within the human plaque biofilm. Previous studies revealed that the iron-binding protein Dpr confers protection toward oxidative stress by limiting free iron availability, which is associated with the generation of toxic hydroxyl radicals. Here, we report the identification of spontaneously occurring mutations within Δdpr strains. Several of those mutations were mapped to the operon smu995-smu998, revealing a previously uncharacterized system that appears to be important in iron acquisition. Disruption of the smu995-smu998 operon resulted in reversion of the stress-sensitive phenotype typical of a Δdpr strain. Our data suggest that the Smu995-Smu998 system works along with other known metal transport systems of S. mutans, i.e., FeoABC and SloABC, to coordinate iron uptake.

A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism.

The exact mechanism underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) associated with the GGGGCC repeat expansion in C9orf72 is still unclear. Two gain-of-function mechanisms are possible: repeat RNA toxicity and dipeptide repeat protein (DPR) toxicity. We here dissected both possibilities using a zebrafish model for ALS. Expression of two DPRs, glycine-arginine and proline-arginine, induced a motor axonopathy. Similarly, expanded sense and antisense repeat RNA also induced a motor axonopathy and formed mainly cytoplasmic RNA foci. However, DPRs were not detected in these conditions. Moreover, stop codon-interrupted repeat RNA still induced a motor axonopathy and a synergistic role of low levels of DPRs was excluded. Altogether, these results show that repeat RNA toxicity is independent of DPR formation. This RNA toxicity, but not the DPR toxicity, was attenuated by the RNA-binding protein Pur-alpha and the autophagy-related protein p62. Our findings demonstrate that RNA toxicity, independent of DPR toxicity, can contribute to the pathogenesis of C9orf72-associated ALS/FTD.

The Wnt Signaling Antagonist Dapper1 Accelerates Dishevelled2 Degradation via Promoting Its Ubiquitination and Aggregate-induced Autophagy.

Autophagy is a regulated process that sequesters and transports cytoplasmic materials such as protein aggregates via autophagosomes to lysosomes for degradation. Dapper1 (Dpr1), an interacting protein of Dishevelled (Dvl), antagonizes Wnt signaling by promoting Dishevelled degradation via lysosomes. However, the mechanism is unclear. Here, we show that Dpr1 promotes the von Hippel-Lindau tumor suppressor (VHL)-mediated ubiquitination of Dvl2 and its autophagic degradation. Knockdown of Dpr1 decreases the interaction between Dvl2 and pVHL, resulting in reduced ubiquitination of Dvl2. Dpr1-mediated autophagic degradation of Dvl2 depends on Dvl2 aggregation. Moreover, the aggregate-prone proteins Dvl2, p62, and the huntingtin mutant Htt103Q promote autophagy in a Dpr1-dependent manner. These protein aggregates enhance the Beclin1-Vps34 interaction and Atg14L puncta formation, indicating that aggregated proteins stimulate autophagy initiation. Ubiquitination is not essential for the aggregate-induced autophagy initiation as inhibition of the ubiquitin-activation E1 enzyme activity did not block the aggregate-induced Atg14L puncta formation. Our findings suggest that Dpr1 promotes the ubiquitination of Dvl2 by pVHL and mediates the protein aggregate-elicited autophagy initiation.