The absence of the ribosomal protein Rpl2702 elicits the MAPK-mTOR signaling to modulate mitochondrial morphology and functions.

Ribosomes mediate protein synthesis, which is one of the most energy-demanding activities within the cell, and mitochondria are one of the main sources generating energy. How mitochondrial morphology and functions are adjusted to cope with ribosomal defects, which can impair protein synthesis and affect cell viability, is poorly understood. Here, we used the fission yeast Schizosaccharomyces Pombe as a model organism to investigate the interplay between ribosome and mitochondria. We found that a ribosomal insult, caused by the absence of Rpl2702, activates a signaling pathway involving Sty1/MAPK and mTOR to modulate mitochondrial morphology and functions. Specifically, we demonstrated that Sty1/MAPK induces mitochondrial fragmentation in a mTOR-independent manner while both Sty1/MAPK and mTOR increases the levels of mitochondrial membrane potential and mitochondrial reactive oxygen species (mROS). Moreover, we demonstrated that Sty1/MAPK acts upstream of Tor1/TORC2 and Tor1/TORC2 and is required to activate Tor2/TORC1. The enhancements of mitochondrial membrane potential and mROS function to promote proliferation of cells bearing ribosomal defects. Hence, our study reveals a previously uncharacterized Sty1/MAPK-mTOR signaling axis that regulates mitochondrial morphology and functions in response to ribosomal insults and provides new insights into the molecular and physiological adaptations of cells to impaired protein synthesis.

A KDELR-mediated ER-retrieval system modulates mitochondrial functions via the unfolded protein response in fission yeast.

KDELR (Erd2 [ER retention defective 2] in yeasts) is a receptor protein that retrieves endoplasmic reticulum (ER)-resident proteins from the Golgi apparatus. However, the role of the KDELR-mediated ER-retrieval system in regulating cellular homeostasis remains elusive. Here, we show that the absence of Erd2 triggers the unfolded protein response (UPR) and enhances mitochondrial respiration and reactive oxygen species in an UPR-dependent manner in the fission yeast Schizosaccharomyces pombe. Moreover, we perform transcriptomic analysis and find that the expression of genes related to mitochondrial respiration and the tricarboxylic acid cycle is upregulated in a UPR-dependent manner in cells lacking Erd2. The increased mitochondrial respiration and reactive oxygen species production is required for cell survival in the absence of Erd2. Therefore, our findings reveal a novel role of the KDELR-Erd2-mediated ER-retrieval system in modulating mitochondrial functions and highlight its importance for cellular homeostasis in the fission yeast.

A multiple-step screening protocol to identify norepinephrine and dopamine reuptake inhibitors for depression.

Depression severely impairs the health of people all over the world. Cognitive dysfunction due to depression has resulted in a severe economic burden to family and society induced by the reduction of social functioning of patients. Norepinephrine-dopamine reuptake inhibitors (NDRIs) targeted with the human norepinephrine transporter (hNET) and distributed with the human dopamine transporter (hDAT) simultaneously treat depression and improve cognitive function, and they effectively prevent sexual dysfunction and other side effects. Because many patients continue to poorly respond to NDRIs, it is urgent to discover novel NDRI antidepressants that do not interfere with cognitive function. The aim of this work was to selectively identify novel NDRI candidates acting against hNET and hDAT from large compound libraries by a comprehensive strategy integrating support vector machine (SVM) models, ADMET, molecular docking, in vitro binding assays, molecular dynamics simulation, and binding energy calculation. First, 6522 compounds that do not inhibit the human serotonin transporter (hSERT) were obtained by SVM models of hNET, hDAT, and non-target hSERT with similarity analyses from compound libraries. ADMET and molecular docking were then used to identify compounds that could potently bind to the hNET and hDAT with satisfactory ADMET, and 4 compounds were successfully identified. According to their docking scores and ADMET information, 3719810 was advanced for profiling by in vitro assays as a novel NDRI lead compound due to its strongest druggability and balancing activities. Encouragingly, 3719810 performed comparative activities on two targets, with Ki values of 7.32 µM for hNET and 5.23 µM for hDAT. To obtain candidates with additional activities and balance the activities of 2 targets, 5 analogs were optimized, and 2 novel scaffold compounds were successively designed. By assessment of molecular docking, molecular dynamics simulations, and binding energy calculations, 5 compounds were validated as NDRI candidates with high activities, and 4 of them performed acceptable balancing activities acting on hNET and hDAT. This work supplied promising novel NDRIs for treatment of depression with cognitive dysfunction or other related neurodegenerative disorders, and also provided a strategy for highly efficient and cost-effective identification of inhibitors for dual targets with homologous non-targets.

Computational Methods for Understanding the Selectivity and Signal Transduction Mechanism of Aminomethyl Tetrahydronaphthalene to Opioid Receptors.

Opioid receptors are members of the group of G protein-couple receptors, which have been proven to be effective targets for treating severe pain. The interactions between the opioid receptors and corresponding ligands and the receptor's activation by different agonists have been among the most important fields in opioid research. In this study, with compound M1, an active metabolite of tramadol, as the clue compound, several aminomethyl tetrahydronaphthalenes were designed, synthesized and assayed upon opioid receptors. With the resultant compounds FW-AII-OH-1 (Ki = 141.2 nM for the κ opioid receptor), FW-AII-OH-2 (Ki = 4.64 nM for the δ opioid receptor), FW-DI-OH-2 (Ki = 8.65 nM for the δ opioid receptor) and FW-DIII-OH-2 (Ki = 228.45 nM for the δ opioid receptor) as probe molecules, the structural determinants responsible for the subtype selectivity and activation mechanisms were further investigated by molecular modeling and molecular dynamics simulations. It was shown that Y7.43 was a key residue in determining the selectivity of the three opioid receptors, and W6.58 was essential for the selectivity of the δ opioid receptor. A detailed stepwise discovered agonist-induced signal transduction mechanism of three opioid receptors by aminomethyl tetrahydronaphthalene compounds was proposed: the 3-7 lock between TM3 and TM7, the DRG lock between TM3 and TM6 and rearrangement of I3.40, P5.50 and F6.44, which resulted in the cooperative movement in 7 TMs. Then, the structural relaxation left room for the binding of the G protein at the intracellular site, and finally the opioid receptors were activated.

Mitochondrial fusion and fission are required for proper mitochondrial function and cell proliferation in fission yeast.

Mitochondria form a branched tubular network in many types of cells, depending on a balance between mitochondrial fusion and fission. How mitochondrial fusion and fission are involved in regulating mitochondrial function and cell proliferation is not well understood. Here, we dissected the roles of mitochondrial fusion and fission in mitochondrial function and cell proliferation in fission yeast. We examined mitochondrial membrane potential by staining cells with DiOC6 and assessed mitochondrial respiration by directly measuring oxygen consumption of cells with a dissolved oxygen respirometer. We found that defects in mitochondrial fission or fusion reduce mitochondrial membrane potential and compromise mitochondrial respiration while the absence of both mitochondrial fusion and fission restores wild type-like respiration, normal membrane potential, and tubular networks of mitochondria. Moreover, we found that the absence of either mitochondrial fission or fusion prolongs the cell cycle and that the absence of both mitochondrial fusion and fission significantly delays cell cycle progression after nitrogen replenishment. The prolonged/delayed cell cycle is likely due to the deregulation of Cdc2 activation. Hence, our work not only establishes an intimate link between mitochondrial morphology and function but also underscores the importance of mitochondrial dynamics in regulating the cell cycle.

Development and evaluation of a deep neural network for histologic classification of renal cell carcinoma on biopsy and surgical resection slides.

Renal cell carcinoma (RCC) is the most common renal cancer in adults. The histopathologic classification of RCC is essential for diagnosis, prognosis, and management of patients. Reorganization and classification of complex histologic patterns of RCC on biopsy and surgical resection slides under a microscope remains a heavily specialized, error-prone, and time-consuming task for pathologists. In this study, we developed a deep neural network model that can accurately classify digitized surgical resection slides and biopsy slides into five related classes: clear cell RCC, papillary RCC, chromophobe RCC, renal oncocytoma, and normal. In addition to the whole-slide classification pipeline, we visualized the identified indicative regions and features on slides for classification by reprocessing patch-level classification results to ensure the explainability of our diagnostic model. We evaluated our model on independent test sets of 78 surgical resection whole slides and 79 biopsy slides from our tertiary medical institution, and 917 surgical resection slides from The Cancer Genome Atlas (TCGA) database. The average area under the curve (AUC) of our classifier on the internal resection slides, internal biopsy slides, and external TCGA slides is 0.98 (95% confidence interval (CI): 0.97-1.00), 0.98 (95% CI: 0.96-1.00) and 0.97 (95% CI: 0.96-0.98), respectively. Our results suggest that the high generalizability of our approach across different data sources and specimen types. More importantly, our model has the potential to assist pathologists by (1) automatically pre-screening slides to reduce false-negative cases, (2) highlighting regions of importance on digitized slides to accelerate diagnosis, and (3) providing objective and accurate diagnosis as the second opinion.

Qualitatively and quantitatively evaluating harm-reduction goal setting among chronically homeless individuals with alcohol dependence.

INTRODUCTION: Most treatment programs for alcohol dependence have prioritized alcohol abstinence as the primary treatment goal. However, abstinence-based goals are not always considered desirable or attainable by more severely affected populations, such as chronically homeless people with alcohol dependence. Because these individuals comprise a multimorbid and high-utilizing population, they are in need of more focused research attention that elucidates their preferred treatment goals. The aim of this secondary study was therefore to qualitatively and quantitatively document participant-generated treatment goals METHODS: Participants were currently or formerly chronically homeless individuals (N=31) with alcohol dependence who participated in a pilot of extended-release naltrexone and harm-reduction counseling. Throughout the treatment period, study interventionists elicited participants' goals and recorded them on an open-ended grid. In subsequent weeks, progress towards and achievement of goals was obtained via self-report and recorded by study interventionists. Conventional content analysis was performed to classify participant-generated treatment goals RESULTS: Representation of the three top categories remained stable over the course of treatment. In the order of their frequency, they included drinking-related goals, quality-of-life goals and health-related goals. Within the category of drinking-related goals, participants consistently endorsed reducing drinking and alcohol-related consequences ahead of abstinence-based goals. Quantitative analyses indicated participants generated an increasing number of goals over the course of treatment. Proportions of goals achieved and progressed towards kept pace with this increase CONCLUSIONS: Findings confirmed hypotheses that chronically homeless people with alcohol dependence can independently generate and achieve treatment goals towards alcohol harm reduction and quality-of-life improvement.