Naoxintong capsule accelerates mitophagy in cerebral ischemia-reperfusion injury via TP53/PINK1/PRKN pathway based on network pharmacology analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.

Pseudomonas aeruginosa Mediates Host Necroptosis through Rhl-Pqs Quorum Sensing Interaction.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that can cause serious infections in immunocompromised patients. Quorum sensing (QS), a communication system evolved by P. aeruginosa to survey its density, is well acknowledged to be involved in various activities during bacterial infection. Recent studies have revealed the link between P. aeruginosa QS and host innate immune response. Previous evidence suggests that programmed cell death exists in response to P. aeruginosa infection. However, it remains unclear whether QS plays a role in the host programmed cell death process during the infection. In this study, we found that the deficiency of one of QS subsystems, rhl, markedly increased mouse bone marrow macrophage cell death induced by P. aeruginosa, which was accompanied by elevated phosphorylation of RIPK3 and MLKL. This highly increased necroptosis activation was caused by the upregulation of another QS subsystem, pqs, because the deletion of pqs in rhl-deficient P. aeruginosa abolished macrophage necroptosis in vitro and in vivo. In sum, our data highlight the cross-talk between P. aeruginosa QS and host necroptosis, which is executed through the rhl-pqs axis.

Significance of Necroptosis in Cartilage Degeneration.

Cartilage, a critical tissue for joint function, often degenerates due to osteoarthritis (OA), rheumatoid arthritis (RA), and trauma. Recent research underscores necroptosis, a regulated form of necrosis, as a key player in cartilage degradation. Unlike apoptosis, necroptosis triggers robust inflammatory responses, exacerbating tissue damage. Key mediators such as receptor-interacting serine/threonine-protein kinase-1 (RIPK1), receptor-interacting serine/threonine-protein kinase-3(RIPK3), and mixed lineage kinase domain-like (MLKL) are pivotal in this process. Studies reveal necroptosis contributes significantly to OA and RA pathophysiology, where elevated RIPK3 and associated proteins drive cartilage degradation. Targeting necroptotic pathways shows promise; inhibitors like Necrostatin-1 (Nec-1), GSK'872, and Necrosulfonamide (NSA) reduce necroptotic cell death, offering potential therapeutic avenues. Additionally, autophagy's role in mitigating necroptosis-induced damage highlights the need for comprehensive strategies addressing multiple pathways. Despite these insights, further research is essential to fully understand necroptosis' mechanisms and develop effective treatments. This review synthesizes current knowledge on necroptosis in cartilage degeneration, aiming to inform novel therapeutic approaches for OA, RA, and trauma.

High fluoride aggravates cadmium-mediated nephrotoxicity of renal tubular epithelial cells through ROS-PINK1/Parkin pathway.

Fluoride (F) and cadmium (Cd) as well known environmental pollutants can cause nephrotoxicity to damage human health, while the joint toxicity of F and Cd to the renal tubular epithelial cells remains still elusive. The interactive influence between F and Cd in oxidative stress, apoptosis, and mitochondrial autophagy of renal tubular epithelial cells was explored. Cells were submitted to varying concentrations with of NaF (1, 5, 10, and 15 µg/mL) combined with CdCl2·2.5H2O (1 µg/mL) for 12 h. Following this, the combined cytotoxicity was assessed. Our results show that different doses of F had varying effects on Cd-mediated nephrotoxicity, with a synergistic effect observed in the high F (15 µg/mL) co-treated with Cd. In response to the Cd induction, the high F treatment resulted in the formation of multiple autophagosomes and notably increased the levels of LDH, ROS, and MMP. It also elevated the MDA contents while decreasing the activities of SOD, GSH-Px, and CAT. Additionally, it yielded a higher Bax/Bcl-2 ratio, which further promotes the apoptotic process. The treatment also disturbed energy metabolism, resulting in a reduction of both ATP and ADP. Furthermore, autophagy-related genes and proteins, including PINK1, Parkin, LC3A, LC3B, and SQSTM1, were significantly improved. In brief, high F of 15 µg/mL aggravated Cd-mediated nephrotoxicity of renal tubular epithelial cells via the ROS-PINK1/Parkin pathway.

Structure and distribution of sensor histidine kinases in the fungal kingdom.

Two-component systems (TCSs) are diverse cell signaling pathways that play a significant role in coping with a wide range of environmental cues in both prokaryotic and eukaryotic organisms. These transduction circuitries are primarily governed by histidine kinases (HKs), which act as sensing proteins of a broad variety of stressors. To date, nineteen HK groups have been previously described in the fungal kingdom. However, the structure and distribution of these prominent sensing proteins were hitherto investigated in a limited number of fungal species. In this study, we took advantage of recent genomic resources in fungi to refine the fungal HK classification by deciphering the structural diversity and phylogenetic distribution of HKs across a large number of fungal clades. To this end, we browsed the genome of 91 species representative of different fungal clades, which yielded 726 predicted HK sequences. A domain organization analysis, coupled with a robust phylogenomic approach, led to an improved categorization of fungal HKs. While most of the compiled sequences were categorized into previously described fungal HK groups, some new groups were also defined. Overall, this study provides an improved overview of the structure, distribution, and evolution of HKs in the fungal kingdom.

CDPK protein in cotton: genomic-wide identification, expression analysis, and conferring resistance to heat stress.

BACKGROUND: Calcium-dependent protein kinase (CDPK) plays a key role in cotton tolerance to abiotic stress. However, its role in cotton heat stress tolerance is not well understood. Here, we characterize the GhCDPK gene family and their expression profiles with the aim of identifying CDPK genes associated with heat stress tolerance. RESULTS: This study revealed 48 GhCDPK members in the cotton genome, distributed on 18 chromosomes. Tree phylogenetic analysis showed three main clustering groups of the GhCDPKs. Cis-elements revealed many abiotic stress and phytohormone pathways conserved promoter regions. Similarly, analysis of the transcription factor binding sites (TFBDS) in the GhCDPK genes showed many stress and hormone related sites. The expression analysis based on qRT-PCR showed that GhCDPK16 was highly responsive to high-temperature stress. Subsequent protein-protein interactions of GhCDPK16 revealed predictable interaction with ROS generating, calcium binding, and ABA signaling proteins. Overexpression of GhCDPK16 in cotton and Arabidopsis improved thermotolerance by lowering ROS compound buildup. Under heat stress, GhCDPK16 transgenic lines upregulated heat-inducible genes GhHSP70, GHSP17.3, and GhGR1, as demonstrated by qRT-PCR analysis. Contrarily, GhCDPK16 knockout lines in cotton exhibited an increase in ROS accumulation. Furthermore, antioxidant enzyme activity was dramatically boosted in the GhCDPK16-ox transgenic lines. CONCLUSIONS: The collective findings demonstrated that GhCDPK16 could be a viable gene to enhance thermotolerance in cotton and, therefore, a potential candidate gene for improving heat tolerance in cotton.

An Inducible Luminescent System to Explore Parkinson's Disease-Associated Genes.

With emerging genetic association studies, new genes and pathways are revealed as causative factors in the development of Parkinson's disease (PD). However, many of these PD genes are poorly characterized in terms of their function, subcellular localization, and interaction with other components in cellular pathways. This represents a major obstacle towards a better understanding of the molecular causes of PD, with deeper molecular studies often hindered by a lack of high-quality, validated antibodies for detecting the corresponding proteins of interest. In this study, we leveraged the nanoluciferase-derived LgBiT-HiBiT system by generating a cohort of tagged PD genes in both induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells. To promote luminescence signals within cells, a master iPSC line was generated, in which LgBiT expression is under the control of a doxycycline-inducible promoter. LgBiT could bind to HiBiT when present either alone or when tagged onto different PD-associated proteins encoded by the genes GBA1, GPNMB, LRRK2, PINK1, PRKN, SNCA, VPS13C, and VPS35. Several HiBiT-tagged proteins could already generate luminescence in iPSCs in response to the doxycycline induction of LgBiT, with the enzyme glucosylceramidase beta 1 (GCase), encoded by GBA1, being one such example. Moreover, the GCase chaperone ambroxol elicited an increase in the luminescence signal in HiBiT-tagged GBA1 cells, correlating with an increase in the levels of GCase in dopaminergic cells. Taken together, we have developed and validated a Doxycycline-inducible luminescence system to serve as a sensitive assay for the quantification, localization, and activity of HiBiT-tagged PD-associated proteins with reliable sensitivity and efficiency.

Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W.

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.

The brassinosteroid receptor gene BRI1 safeguards cell-autonomous brassinosteroid signaling across tissues.

Brassinosteroid signaling is essential for plant growth as exemplified by the dwarf phenotype of loss-of-function mutants in BRASSINOSTEROID INSENSITIVE 1 (BRI1), a ubiquitously expressed Arabidopsis brassinosteroid receptor gene. Complementation of brassinosteroid-blind receptor mutants by BRI1 expression with various tissue-specific promoters implied that local brassinosteroid signaling may instruct growth non-cell autonomously. Here, we performed such rescues with a panel of receptor variants and promoters, in combination with tissue-specific transgene knockouts. Our experiments demonstrate that brassinosteroid receptor expression in several tissues is necessary but not sufficient for rescue. Moreover, complementation with tissue-specific promoters requires the genuine BRI1 gene body sequence, which confers ubiquitous expression of trace receptor amounts that are sufficient to promote brassinosteroid-dependent root growth. Our data, therefore, argue for a largely cell-autonomous action of brassinosteroid receptors.

Prognostic significance and response to immune checkpoint inhibitors of RIPK3, MLKL and necroptosis in non-small cell lung cancer.

Lung cancer remains the leading cause of cancer death. Treatment with immune checkpoint inhibitor (ICI) alone or combination with chemotherapy served as first-line therapy in non-small cell lung cancer (NSCLC). However, only 20-50% of NSCLC patients respond to ICI. Necroptosis, an inflammatory form of cell death plays an important role in the regulation of tumor immune microenvironment which may affect prognosis and ICI response but its clinical significance in NSCLC patients has remained largely unknown. Therefore, we aimed to analyze the correlation between key necroptotic proteins and necroptosis and clinical outcomes, the status of tumor-infiltrating immune cells, and response to ICI in NSCLC patients. The expression of receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) were immunolocalized in 125 surgically resected NSCLC patients and 23 NSCLC patients administered with ICI therapy. CD8 + and FOXp3 + T cells and CD163 + M2 macrophages were also immunolocalized. High RIPK3 status was positively correlated with survival of the patients and RIPK3 turned out an independent favorable prognostic factor of the patients. RIPK3 was negatively correlated with CD8 + T cells, while MLKL positively correlated with CD163 + M2 macrophages, suggesting the possible involvement of RIPK3 and MLKL in formulating immunosuppressive microenvironment. In addition, high RIPK3 status tended to be associated with clinical resistance to ICI therapy (P-value = 0.057). Furthermore, NSCLC cells-expressing RIPK3 suppressed T cells response to ICI therapy in vitro. Therefore, RIPK3 and MLKL could induce an immunosuppressive microenvironment, resulting in low response to ICI therapy in NSCLC.

Activation of parkin by a molecular glue.

Mutations in parkin and PINK1 cause early-onset Parkinson's disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson's disease.

Empowering AlphaFold2 for protein conformation selective drug discovery with AlphaFold2-RAVE.

Small-molecule drug design hinges on obtaining co-crystallized ligand-protein structures. Despite AlphaFold2's strides in protein native structure prediction, its focus on apo structures overlooks ligands and associated holo structures. Moreover, designing selective drugs often benefits from the targeting of diverse metastable conformations. Therefore, direct application of AlphaFold2 models in virtual screening and drug discovery remains tentative. Here, we demonstrate an AlphaFold2-based framework combined with all-atom enhanced sampling molecular dynamics and Induced Fit docking, named AF2RAVE-Glide, to conduct computational model-based small-molecule binding of metastable protein kinase conformations, initiated from protein sequences. We demonstrate the AF2RAVE-Glide workflow on three different mammalian protein kinases and their type I and II inhibitors, with special emphasis on binding of known type II kinase inhibitors which target the metastable classical DFG-out state. These states are not easy to sample from AlphaFold2. Here, we demonstrate how with AF2RAVE these metastable conformations can be sampled for different kinases with high enough accuracy to enable subsequent docking of known type II kinase inhibitors with more than 50% success rates across docking calculations. We believe the protocol should be deployable for other kinases and more proteins generally.

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-ß activation. Activated PKC-ß induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

Kinome state is predictive of cell viability in pancreatic cancer tumor and cancer-associated fibroblast cell lines.

Numerous aspects of cellular signaling are regulated by the kinome-the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation driving many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. In this work, we attempt to link the state of the human kinome, or kinotype, with cell viability in treated, patient-derived PDAC tumor and cancer-associated fibroblast cell lines. We applied classification models to independent kinome perturbation and kinase inhibitor cell screen data, and found that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines, including the understudied kinases CSNK2A1/3, CAMKK2, and PIP4K2C. We next utilized these models to predict the response of new, clinical kinase inhibitors that were not present in the initial dataset for model devlopment and conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the perturbed state of the human protein kinome provides significant opportunity for better understanding of signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapeutic strategies for PDAC.

Cardioprotective effect of Vitamin D on cardiac hypertrophy through improvement of mitophagy and apoptosis in an experimental rat model of levothyroxine -induced hyperthyroidism.

BACKGROUND: Mitochondria are known to be involved in mediating the calorigenic effects of thyroid hormones. With an abundance of these hormones, alterations in energy metabolism and cellular respiration take place, leading to the development of cardiac hypertrophy. Vitamin D has recently gained attention due to its involvement in the regulation of mitochondrial function, demonstrating promising potential in preserving the integrity and functionality of the mitochondrial network. The present study aimed to investigate the therapeutic potential of Vitamin D on cardiac hypertrophy induced by hyperthyroidism, with a focus on the contributions of mitophagy and apoptosis as possible underlying molecular mechanisms. METHODS AND RESULTS: The rats were divided into three groups: control; hyperthyroid; hyperthyroid + Vitamin D. Hyperthyroidism was induced by Levothyroxine administration for four weeks. Serum thyroid hormones levels, myocardial damage markers, cardiac hypertrophy indices, and histological examination were assessed. The assessment of Malondialdehyde (MDA) levels and the expression of the related genes were conducted using heart tissue samples. Vitamin D pretreatment exhibited a significant improvement in the hyperthyroidism-induced decline in markers indicative of myocardial damage, oxidative stress, and indices of cardiac hypertrophy. Vitamin D pretreatment also improved the downregulation observed in myocardial expression levels of genes involved in the regulation of mitophagy and apoptosis, including PTEN putative kinase 1 (PINK1), Mitofusin-2 (MFN2), Dynamin-related Protein 1 (DRP1), and B cell lymphoma-2 (Bcl-2), induced by hyperthyroidism. CONCLUSIONS: These results suggest that supplementation with Vitamin D could be advantageous in preventing the progression of cardiac hypertrophy and myocardial damage.

A secreted fungal laccase targets the receptor kinase OsSRF3 to inhibit OsBAK1-OsSRF3-mediated immunity in rice.

The identification effector targets and characterization of their functions are crucial for understanding pathogen infection mechanisms and components of plant immunity. Here, we identify the effector UgsL, a ustilaginoidin synthetase with a key role in regulating virulence of the rice false smut fungus Ustilaginoidea virens. Heterologous expression of UgsL in rice (Oryza sativa) enhances plant susceptibility to multiple pathogens, and host-induced gene silencing of UgsL enhances plant resistance to U. virens, indicating that UgsL inhibits rice immunity. UgsL interacts with STRUBBELIG RECEPTOR KINASE 3 (OsSRF3). Genome editing and overexpression of OsSRF3 demonstrate that OsSRF3 plays a pivotal role in the resistance of rice to multiple pathogens. Remarkably, overexpressing OsSRF3 enhances resistance without adversely affecting plant growth or yield. We show that BRASSINOSTEROID RECEPTOR-ASSOCIATED KINASE 1 (OsBAK1) interacts with and phosphorylates OsSRF3 to activate pathogen-triggered immunity, inducing the mitogen-activated protein kinase cascade, a reactive oxygen species burst, callose deposition, and expression of defense-related genes. UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1. Furthermore, UgsL mediates OsSRF3 degradation by facilitating its association with the ubiquitin-26S proteasome. Our results reveal that OsSRF3 positively regulates immunity in rice and that UgsL mediates its degradation, thereby inhibiting the activation of OsBAK1-OsSRF3-mediated immune pathways.

[Clinical and genetic analysis of a child with co-morbid progressive IgA nephropathy and COQ8B-associated glomerulopathy].

OBJECTIVE: To explore the genetic etiology and clinical outcome of a child with co-morbid progressive IgA nephropathy and COQ8B-associated glomerulopathy. METHODS: A child who was admitted to Peking University First Hospital on March 2, 2021 was selected as the study subject. Genomic DNA was extracted from peripheral blood samples from the child and his parents and sister. Whole exome sequencing was carried out, and candidate variant was verified by Sanger sequencing. This study was approved by the Peking University First Hospital (Ethics No. 2016[1029]). RESULTS: The child, a 7-year-old boy who had developed proteinuria 8 months before, was diagnosed with IgA nephropathy (M1E1S1T1C1). With steroid, cyclophosphamide, cyclosporine and angiotensin-converting enzyme inhibitor therapy, partial remission of proteinuria was achieved. However, his serum creatinine level had increased from 53.8 mol/L at the onset of disease to 86.7 mol/L after 3.9 years, along with massive proteinuria. Kidney biopsy still indicated IgA nephropathy (M0E0S1T0C0). The child was found to harbor a homozygous c.737G>A (p.Ser246Asn) missense variant of the COQ8B gene, for which his parents and sister were heterozygous carriers. The variant was predicted to be pathogenic (PS1+PM2_Supporting+PM3+PP3+PP4) based on the guidelines from the American College of Medical Genetics and Genomics. The child was treated with high-dose coenzyme Q10 in combination with steroid and/or mycophenolate mofetil, though his serum creatinine level still increased to 286 mol/L after 7.3 years, which conformed to a chronic kidney disorder with glomerular filtration rate category of G3b. CONCLUSION: The homozygous c.737G>A missense variants of the COQ8B gene probably underlay the progressive kidney dysfunction in this child. For children with IgA nephropathy presenting with atypical clinical manifestations, unsatisfactory therapeutic effect, and/or early onset of kidney function decline, coexistence of other diseases should be suspected.

PINK1 insufficiency can be exploited as a specific target for drug combinations inducing mitochondrial pathology-mediated cell death in gastric adenocarcinoma.

There exist very limited non-hazardous therapeutic strategies except for surgical resection and lymphadenectomy against gastric cancer (GC) despite being the third leading cause of cancer deaths worldwide. This study proposes an innovative treatment approach against GC using a drug combination strategy that manipulates mitochondrial dynamics in conjunction with the induction of mitochondrial pathology-mediated cell death. Comparative analysis was done with gastric adenocarcinoma and normal cells by qPCR, western blot, microscopic immunocytochemistry, and live cell imaging. In this study, impairment of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission by Mdivi-1 created an imbalance in mitochondrial structural dynamics in indomethacin-treated AGS cells in which mitophagy-regulator protein PINK1 is downregulated. These drug combinations with the individual sub-lethal doses ultimately led to the activation of cell death machinery upregulating pro-apoptotic proteins like Bax, Puma, and Noxa. Interestingly, this combinatorial therapy did not affect normal gastric epithelial cells significantly and also no significant upregulation of death markers was observed. Moreover, the drug combination strategy also retarded cell migration and reduced stemness in GC cells. In summary, this study offers a pioneering specific therapeutic strategy for GC treatment, sparing normal cells providing opportunities for minimal drug-mediated toxicity utilizing mitochondria as a viable and specific target for anti-cancer therapy in gastric cancer.