Integrated genome-wide association and transcriptomic analysis to identify receptor kinase genes to stripe rust resistance in wheat germplasm from southwestern China.

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. Identification of new and elite Pst-resistance loci or genes has the potential to enhance overall resistance to this pathogen. Here, we conducted an integrated genome-wide association study (GWAS) and transcriptomic analysis to screen for loci associated with resistance to stripe rust in 335 accessions from Yunnan, including 311 landraces and 24 cultivars. Based on the environmental phenotype, we identified 113 protein kinases significantly associated with Pst resistance using mixed linear model (MLM) and generalized linear model (GLM) models. Transcriptomic analysis revealed that 52 of 113 protein kinases identified by GWAS were up and down regulated in response to Pst infection. Among these genes, a total of 15 receptor kinase genes were identified associated with Pst resistance. 11 candidate genes were newly discovered in Yunnan wheat germplasm. Our results revealed that resistance alleles to stripe rust were accumulated in Yunnan wheat germplasm, implying direct or indirect selection for improving stripe rust resistance in elite wheat breeding programs.

Mono-quinoxaline-induced DNA structural alteration leads to ZBP1/RIP3/MLKL-driven necroptosis in cancer cells.

Evading the cellular apoptosis mechanism by modulating multiple pathways poses a sturdy barrier to effective chemotherapy. Cancer cell adeptly resists the apoptosis signaling pathway by regulating anti and pro-apoptotic proteins to escape cell death. Nevertheless, bypassing the apoptotic pathway through necroptosis, an alternative programmed cell death process, maybe a potential therapeutic modality for apoptosis-resistant cells. However, synthetic mono-quinoxaline-based intercalator-induced cellular necroptosis as an anti-cancer perspective remains under-explored. To address this concern, we undertook the design and synthesis of quinoxaline-based small molecules (3a-3l). Our approach involved enhancing the π-surface of the mandatory benzyl moiety to augment its ability to induce DNA structural alteration via intercalation, thereby promoting cytotoxicity across various cancer cell lines (HCT116, HT-29, and HeLa). Notably, the potent compound 3a demonstrated the capacity to induce DNA damage in cancer cells, leading to the induction of ZBP1-mediated necroptosis in the RIP3-expressed cell line (HT-29), where Z-VAD effectively blocked apoptosis-mediated cell death. Interestingly, we observed that 3a induced RIP3-driven necroptosis in combination with DNA hypomethylating agents, even in the RIP3-silenced cell lines (HeLa and HCT116). Overall, our synthesized compound 3a emerged as a promising candidate against various cancers, particularly in apoptosis-compromised cells, through the induction of necroptosis.

Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

Identification and structural characterization of small molecule inhibitors of PINK1.

Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

The mitochondria-targeted Kaempferol nanoparticle ameliorates severe acute pancreatitis.

Kaempferol (KA), an natural antioxidant of traditional Chinese medicine (TCM), is extensively used as the primary treatment for inflammatory digestive diseases with impaired redox homeostasis. Severe acute pancreatitis (SAP) was exacerbated by mitochondrial dysfunction and abundant ROS, which highlights the role of antioxidants in targeting mitochondrial function. However, low bioavailability and high dosage of KA leading to unavoidable side effects limits clinical transformation. The mechanisms of KA with poor bioavailability largely unexplored, hindering development of the efficient strategies to maximizing the medicinal effects of KA. Here, we engineered a novel thioketals (TK)-modified based on DSPE-PEG2000 liposomal codelivery system for improving bioavailability and avoiding side effects (denotes as DSPE-TK-PEG2000-KA, DTM@KA NPs). We demonstrated that the liposome exerts profound impacts on damaging intracellular redox homeostasis by reducing GSH depletion and activating Nrf2, which synergizes with KA to reinforce the inhibition of inadequate fission, excessive mitochondrial fusion and impaired mitophagy resulting in inflammation and apoptosis; and then, the restored mitochondrial homeostasis strengthens ATP supply for PAC renovation and homeostasis. Interestingly, TK bond was proved as the main functional structure to improve the above efficacy of KA compared with the absence of TK bond. Most importantly, DTM@KA NPs obviously suppresses PAC death with negligible side effects in vitro and vivo. Mechanismly, DTM@KA NPs facilitated STAT6-regulated mitochondrial precursor proteins transport via interacting with TOM20 to further promote Drp1-dependent fission and Pink1/Parkin-regulated mitophagy with enhanced lysosomal degradation for removing damaged mitochondria in PAC and then reduce inflammation and apoptosis. Generally, DTM@KA NPs synergistically improved mitochondrial homeostasis, redox homeostasis, energy metabolism and inflammation response via regulating TOM20-STAT6-Drp1 signaling and promoting mitophagy in SAP. Consequently, such a TCM's active ingredients-based nanomedicine strategy is be expected to be an innovative approach for SAP therapy.

SIRT3 alleviates painful diabetic neuropathy by mediating the FoxO3a-PINK1-Parkin signaling pathway to activate mitophagy.

INTRODUCTION: Painful diabetic neuropathy (PDN) is a common complication of diabetes. Previous studies have implicated that mitochondrial dysfunction plays a role in the development of PDN, but its pathogenesis and mechanism have not been fully investigated. METHODS: In this study, we used high-fat diet/low-dose streptozotocin-induced rats as a model of type 2 diabetes mellitus. Behavioral testing, whole-cell patch-clamp recordings of dorsal root ganglion (DRG) neurons, and complex sensory nerve conduction velocity studies were used to assess peripheral neuropathy. Mitochondrial membrane potential (MMP), ATP, tissue reactive oxygen species, and transmission electron microscopy were used to evaluate the function and morphology of mitochondria in DRG. Real-time PCR, western blot, and immunofluorescence were performed to investigate the mechanism. RESULTS: We found that damaged mitochondria were accumulated and mitophagy was inhibited in PDN rats. The expression of sirtuin 3 (SIRT3), which is an NAD+-dependent deacetylase in mitochondria, was inhibited. Overexpression of SIRT3 in DRG neurons by intrathecally administered LV-SIRT3 lentivirus ameliorated neurological and mitochondrial dysfunctions. This was evidenced by the reversal of allodynia and nociceptor hyperexcitability, as well as the restoration of MMP and ATP levels. Overexpression of SIRT3 restored the inhibited mitophagy by activating the FoxO3a-PINK1-Parkin signaling pathway. The effects of SIRT3 overexpression, including the reversal of allodynia and nociceptor hyperexcitability, the improvement of impaired mitochondria and mitophagy, and the restoration of PINK1 and Parkin expression, were counteracted when FoxO3a siRNA was intrathecally injected. CONCLUSION: These results showed that SIRT3 overexpression ameliorates PDN via activation of FoxO3a-PINK1-Parkin-mediated mitophagy, suggesting that SIRT3 may become an encouraging therapeutic strategy for PDN.

Panax notoginseng saponins prevent dementia and oxidative stress in brains of SAMP8 mice by enhancing mitophagy.

BACKGROUND: Mitochondrial dysfunction is one of the distinctive features of neurons in patients with Alzheimer's disease (AD). Intraneuronal autophagosomes selectively phagocytose and degrade the damaged mitochondria, mitigating neuronal damage in AD. Panax notoginseng saponins (PNS) can effectively reduce oxidative stress and mitochondrial damage in the brain of animals with AD, but their exact mechanism of action is unknown. METHODS: Senescence-accelerated mouse prone 8 (SAMP8) mice with age-related AD were treated with PNS for 8 weeks. The effects of PNS on learning and memory abilities, cerebral oxidative stress status, and hippocampus ultrastructure of mice were observed. Moreover, changes of the PTEN-induced putative kinase 1 (PINK1)-Parkin, which regulates ubiquitin-dependent mitophagy, and the recruit of downstream autophagy receptors were investigated. RESULTS: PNS attenuated cognitive dysfunction in SAMP8 mice in the Morris water maze test. PNS also enhanced glutathione peroxidase and superoxide dismutase activities, and increased glutathione levels by 25.92% and 45.55% while inhibiting 8-hydroxydeoxyguanosine by 27.74% and the malondialdehyde production by 34.02% in the brains of SAMP8 mice. Our observation revealed the promotion of mitophagy, which was accompanied by an increase in microtubule-associated protein 1 light chain 3 (LC3) mRNA and 70.00% increase of LC3-II/I protein ratio in the brain tissues of PNS-treated mice. PNS treatment increased Parkin mRNA and protein expression by 62.80% and 43.80%, while increasing the mRNA transcription and protein expression of mitophagic receptors such as optineurin, and nuclear dot protein 52. CONCLUSION: PNS enhanced the PINK1/Parkin pathway and facilitated mitophagy in the hippocampus, thereby preventing cerebral oxidative stress in SAMP8 mice. This may be a mechanism contributing to the cognition-improvement effect of PNS.

Structural insights into the regulation of protein-arginine kinase McsB by McsA.

In gram-positive bacteria, phosphorylated arginine functions as a protein degradation signal in a similar manner as ubiquitin in eukaryotes. The protein-arginine phosphorylation is mediated by the McsAB complex, where McsB possesses kinase activity and McsA modulates McsB activity. Although mcsA and mcsB are regulated within the same operon, the role of McsA in kinase activity has not yet been clarified. In this study, we determined the molecular mechanism by which McsA regulates kinase activity. The crystal structure of the McsAB complex shows that McsA binds to the McsB kinase domain through a second zinc-coordination domain and the subsequent loop region. This binding activates McsB kinase activity by rearranging the catalytic site, preventing McsB self-assembly, and enhancing stoichiometric substrate binding. The first zinc-coordination and coiled-coil domains of McsA further activate McsB by reassembling the McsAB oligomer. These results demonstrate that McsA is the regulatory subunit for the reconstitution of the protein-arginine kinase holoenzyme. This study provides structural insight into how protein-arginine kinase directs the cellular protein degradation system.

Effects of carbamazepine on BDNF expression in trigeminal ganglia and serum in rats with trigeminal neuralgia. / å¡é©¬è¥¿å¹³å¯¹ä¸‰å‰ç¥žç»ç—›å¤§é¼ ä¸‰å‰ç¥žç»èŠ‚åŠè¡€æ¸ ä¸­BDNF表达变化的影响.

OBJECTIVES: Trigeminal neuralgia (TN) is a severe chronic neuropathic pain that mainly affects the distribution area of the trigeminal nerve with limited treating efficacy. There are numerous treatments for TN, but currently the main clinical approach is to suppress pain by carbamazepine (CBZ). Brain-derived neurotrophic factor (BDNF) is closely related to chronic pain. This study aims to determine the effects of CBZ treatment on BDNF expression in both the trigeminal ganglion (TG) and serum of TN via a chronic constriction injury of the infraorbital nerve (ION-CCI) rat model. METHODS: The ION-CCI models were established in male Sprague-Dawley rats and were randomly divided into a sham group, a TN group, a TN+low-dose CBZ treatment group (TN+20 mg/kg CBZ group), a TN+medium-dose CBZ treatment group (TN+40 mg/kg CBZ group), and a TN+high-dose CBZ treatment group (TN+80 mg/kg CBZ group). The mechanical pain threshold in each group of rats was measured regularly before and after surgery. The expressions of BDNF and tyrosine kinase receptor B (TrkB) mRNA in TGs of rats in different groups were determined by real-time PCR, and the expression of BDNF protein on neurons in TGs was observed by immunofluorescence. Western Blotting was used to detect the protein expression of BDNF, TrkB, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) in TGs of rats in different groups. The expression of BDNF in the serum of rats in different groups was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of mechanical pain sensitivity showed that there was no significant difference in the mechanical pain threshold in the right facial sensory area of the experimental rats in each group before surgery (all P>0.05). From the 3rd day after operation, the mechanical pain threshold of rats in the TN group was significantly lower than that in the sham group (all P<0.01), and the mechanical pain threshold of rats in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 CBZ mg/kg group was higher than that in the TN group (all P<0.05). The BDNF and TrkB mRNA and protein expressions in TGs of rats in the TN group were higher than those in the sham group (all P<0.05), and those in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than the TN group (all P<0.05). The p-ERK levels in TG of rats in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were significantly decreased compared with the TN group (all P<0.05). The BDNF and neuron-specific nuclear protein (NeuN) were mainly co-expressed in neuron of TGs in the TN group and they were significantly higher than those in the sham group (all P<0.05). The co-labeled expressions of BDNF and NeuN in TGs of the TN+ 80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than those in the TN group (all P<0.05). The results of ELISA showed that the level of BDNF in the serum of the TN group was significantly higher than that in the sham group (P<0.05). The levels of BDNF in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than those in the TN group (all P<0.05). Spearman correlation analysis showed that the BDNF level in serum was negatively correlated with mechanical pain threshold (r=-0.650, P<0.01). CONCLUSIONS: CBZ treatment can inhibit the expression of BDNF and TrkB in the TGs of TN rats, reduce the level of BDNF in serum of TN rats and the phosphorylation of ERK signaling pathway, so as to inhibit TN. The serum level of BDNF can be considered as an indicator for the diagnosis and prognosis of TN.

KdpD is a tandem serine histidine kinase that controls K+ pump KdpFABC transcriptionally and post-translationally.

Two-component systems, consisting of a histidine kinase and a response regulator, serve signal transduction in bacteria, often regulating transcription in response to environmental stimuli. Here, we identify a tandem serine histidine kinase function for KdpD, previously described as a histidine kinase of the KdpDE two-component system, which controls production of the potassium pump KdpFABC. We show that KdpD additionally mediates an inhibitory serine phosphorylation of KdpFABC at high potassium levels, using not its C-terminal histidine kinase domain but an N-terminal atypical serine kinase domain. Sequence analysis of KdpDs from different species highlights that some KdpDs are much shorter than others. We show that, while Escherichia coli KdpD's atypical serine kinase domain responds directly to potassium levels, a shorter version from Deinococcus geothermalis is controlled by second messenger cyclic di-AMP. Our findings add to the growing functional diversity of sensor kinases while simultaneously expanding the framework for regulatory mechanisms in bacterial potassium homeostasis.

Angiotensin II Is Involved in MLKL Activation During the Development of Heart Failure Following Myocardial Infarction in Rats.

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT1 receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

Luteolin-7-O-ß-d-glucuronide Ameliorates Cerebral Ischemic Injury: Involvement of RIP3/MLKL Signaling Pathway.

Luteolin-7-O-ß-d-glucuronide (LGU) is a major active flavonoid glycoside compound that is extracted from Ixeris sonchifolia (Bge.) Hance, and it is a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the neuroprotective effect of LGU was investigated in an oxygen glucose deprivation (OGD) model and a middle cerebral artery occlusion (MCAO) rat model. In vitro, LGU was found to effectively improve the OGD-induced decrease in neuronal viability and increase in neuronal death by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) leakage rate assay, respectively. LGU was also found to inhibit OGD-induced intracellular Ca2+ overload, adenosine triphosphate (ATP) depletion, and mitochondrial membrane potential (MMP) decrease. By Western blotting analysis, LGU significantly inhibited the OGD-induced increase in expressions of receptor-interacting serine/threonine-protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). Moreover, molecular docking analysis showed that LGU might bind to RIP3 more stably and firmly than the RIP3 inhibitor GSK872. Immunofluorescence combined with confocal laser analyses disclosed that LGU inhibited the aggregation of MLKL to the nucleus. Our results suggest that LGU ameliorates OGD-induced rat primary cortical neuronal injury via the regulation of the RIP3/MLKL signaling pathway in vitro. In vivo, LGU was proven, for the first time, to protect the cerebral ischemia in a rat middle cerebral artery occlusion (MCAO) model, as shown by improved neurological deficit scores, infarction volume rate, and brain water content rate. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia.

Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

Genetic architecture of the structural connectome.

Myelinated axons form long-range connections that enable rapid communication between distant brain regions, but how genetics governs the strength and organization of these connections remains unclear. We perform genome-wide association studies of 206 structural connectivity measures derived from diffusion magnetic resonance imaging tractography of 26,333 UK Biobank participants, each representing the density of myelinated connections within or between a pair of cortical networks, subcortical structures or cortical hemispheres. We identify 30 independent genome-wide significant variants after Bonferroni correction for the number of measures studied (126 variants at nominal genome-wide significance) implicating genes involved in myelination (SEMA3A), neurite elongation and guidance (NUAK1, STRN, DPYSL2, EPHA3, SEMA3A, HGF, SHTN1), neural cell proliferation and differentiation (GMNC, CELF4, HGF), neuronal migration (CCDC88C), cytoskeletal organization (CTTNBP2, MAPT, DAAM1, MYO16, PLEC), and brain metal transport (SLC39A8). These variants have four broad patterns of spatial association with structural connectivity: some have disproportionately strong associations with corticothalamic connectivity, interhemispheric connectivity, or both, while others are more spatially diffuse. Structural connectivity measures are highly polygenic, with a median of 9.1 percent of common variants estimated to have non-zero effects on each measure, and exhibited signatures of negative selection. Structural connectivity measures have significant genetic correlations with a variety of neuropsychiatric and cognitive traits, indicating that connectivity-altering variants tend to influence brain health and cognitive function. Heritability is enriched in regions with increased chromatin accessibility in adult oligodendrocytes (as well as microglia, inhibitory neurons and astrocytes) and multiple fetal cell types, suggesting that genetic control of structural connectivity is partially mediated by effects on myelination and early brain development. Our results indicate pervasive, pleiotropic, and spatially structured genetic control of white-matter structural connectivity via diverse neurodevelopmental pathways, and support the relevance of this genetic control to healthy brain function.

Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis.

Copine proteins are highly conserved and ubiquitously found in eukaryotes, and their indispensable roles in different species were proposed. However, their exact function remains unclear. The phytohormone brassinosteroids (BRs) play vital roles in plant growth, development and environmental responses. A key event in effective BR signaling is the formation of functional BRI1-SERK receptor complex and subsequent transphosphorylation upon ligand binding. Here, we demonstrate that BONZAI (BON) proteins, which are plasma membrane-associated copine proteins, are critical components of BR signaling in both the monocot maize and the dicot Arabidopsis. Biochemical and molecular analyses reveal that BON proteins directly interact with SERK kinases, thereby ensuring effective BRI1-SERK interaction and transphosphorylation. This study advances the knowledge on BR signaling and provides an important target for optimizing valuable agronomic traits, it also opens a way to study steroid hormone signaling and copine proteins of eukaryotes in a broader perspective.

RN0D, a galactoglucan from Panax notoginseng flower induces cancer cell death via PINK1/Parkin mitophagy.

Metabolic alterations within mitochondria, encompassing processes such as autophagy and energy metabolism, play a pivotal role in facilitating the swift proliferation, invasion, and metastasis of cancer cells. Despite this, there is a scarcity of currently available medications with proven anticancer efficacy through the modulation of mitochondrial dysfunction in a clinical setting. Here, we introduce the structural characteristics of RN0D, a galactoglucan isolated and purified from Panax notoginseng flowers, mainly composed of ß-1,4-galactan and ß-1,3/1,6-glucan. RN0D demonstrates the capacity to induce mitochondrial impairment in cancer cells, leading to the accumulation of reactive oxygen species, initiation of mitophagy, and reduction in both mitochondrial number and size. This sequence of events ultimately results in the inhibition of mitochondrial and glycolytic bioenergetics, culminating in the demise of cancer cells due to adenosine triphosphate (ATP) deprivation. Notably, the observed bioactivity is attributed to RN0D's direct targeting of Galectin-3, as affirmed by surface plasmon resonance studies. Furthermore, RN0D is identified as an activator of the PTEN-induced kinase 1 (PINK1)/Parkin pathway, ultimately instigating cytotoxic mitophagy in tumor cells. This comprehensive study substantiates the rationale for advancing RN0D as a potentially efficacious anticancer therapeutic.

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis.

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.

Cellular and clinical impact of protein phosphatase enzyme epigenetic silencing in multiple cancer tissues.

BACKGROUND: Protein Phosphatase Enzymes (PPE) and protein kinases simultaneously control phosphorylation mechanisms that tightly regulate intracellular signalling pathways and stimulate cellular responses. In human malignancies, PPE and protein kinases are frequently mutated resulting in uncontrolled kinase activity and PPE suppression, leading to cell proliferation, migration and resistance to anti-cancer therapies. Cancer associated DNA hypermethylation at PPE promoters gives rise to transcriptional silencing (epimutations) and is a hallmark of cancer. Despite recent advances in sequencing technologies, data availability and computational capabilities, only a fraction of PPE have been reported as transcriptionally inactive as a consequence of epimutations. METHODS: In this study, we examined promoter-associated DNA methylation profiles in Protein Phosphatase Enzymes and their Interacting Proteins (PPEIP) in a cohort of 705 cancer patients in five tissues (Large intestine, Oesophagus, Lung, Pancreas and Stomach) in three cell models (primary tumours, cancer cell lines and 3D embedded cancer cell cultures). As a subset of PPEIP are known tumour suppressor genes, we analysed the impact of PPEIP promoter hypermethylation marks on gene expression, cellular networks and in a clinical setting. RESULTS: Here, we report epimutations in PPEIP are a frequent occurrence in the cancer genome and manifest independent of transcriptional activity. We observed that different tumours have varying susceptibility to epimutations and identify specific cellular signalling networks that are primarily affected by epimutations. Additionally, RNA-seq analysis showed the negative impact of epimutations on most (not all) Protein Tyrosine Phosphatase transcription. Finally, we detected novel clinical biomarkers that inform on patient mortality and anti-cancer treatment sensitivity. CONCLUSIONS: We propose that DNA hypermethylation marks at PPEIP frequently contribute to the pathogenesis of malignancies and within the precision medicine space, hold promise as biomarkers to inform on clinical features such as patient survival and therapeutic response.

Gasdermin and MLKL necrotic cell death effectors: Signaling and diseases.

Diverse inflammatory conditions, from infections to autoimmune disease, are often associated with cellular damage and death. Apoptotic cell death has evolved to minimize its inflammatory potential. By contrast, necrotic cell death via necroptosis and pyroptosis-driven by membrane-damaging MLKL and gasdermins, respectively-can both initiate and propagate inflammatory responses. In this review, we provide insights into the function and regulation of MLKL and gasdermin necrotic effector proteins and drivers of plasma membrane rupture. We evaluate genetic evidence that MLKL- and gasdermin-driven necrosis may either provide protection against, or contribute to, disease states in a context-dependent manner. These cumulative insights using gene-targeted mice underscore the necessity for future research examining pyroptotic and necroptotic cell death in human tissue, as a basis for developing specific necrotic inhibitors with the potential to benefit a spectrum of pathological conditions.

Rapid translocation of NGR proteins driving polarization of PIN-activating D6 protein kinase during root gravitropism.

Root gravitropic bending represents a fundamental aspect of terrestrial plant physiology. Gravity is perceived by sedimentation of starch-rich plastids (statoliths) to the bottom of the central root cap cells. Following gravity perception, intercellular auxin transport is redirected downwards leading to an asymmetric auxin accumulation at the lower root side causing inhibition of cell expansion, ultimately resulting in downwards bending. How gravity-induced statoliths repositioning is translated into asymmetric auxin distribution remains unclear despite PIN auxin efflux carriers and the Negative Gravitropic Response of roots (NGR) proteins polarize along statolith sedimentation, thus providing a plausible mechanism for auxin flow redirection. In this study, using a functional NGR1-GFP construct, we visualized the NGR1 localization on the statolith surface and plasma membrane (PM) domains in close proximity to the statoliths, correlating with their movements. We determined that NGR1 binding to these PM domains is indispensable for NGR1 functionality and relies on cysteine acylation and adjacent polybasic regions as well as on lipid and sterol PM composition. Detailed timing of the early events following graviperception suggested that both NGR1 repolarization and initial auxin asymmetry precede the visible PIN3 polarization. This discrepancy motivated us to unveil a rapid, NGR-dependent translocation of PIN-activating AGCVIII kinase D6PK towards lower PMs of gravity-perceiving cells, thus providing an attractive model for rapid redirection of auxin fluxes following gravistimulation.