Arabidopsis Sec14 proteins (SFH5 and SFH7) mediate interorganelle transport of phosphatidic acid and regulate chloroplast development.

Lipids establish the specialized thylakoid membrane of chloroplast in eukaryotic photosynthetic organisms, while the molecular basis of lipid transfer from other organelles to chloroplast remains further elucidation. Here we revealed the structural basis of Arabidopsis Sec14 homology proteins AtSFH5 and AtSFH7 in transferring phosphatidic acid (PA) from endoplasmic reticulum (ER) to chloroplast, and whose function in regulating the lipid composition of chloroplast and thylakoid development. AtSFH5 and AtSFH7 localize at both ER and chloroplast, whose deficiency resulted in an abnormal chloroplast structure and a decreased thickness of stacked thylakoid membranes. We demonstrated that AtSFH5, but not yeast and human Sec14 proteins, could specifically recognize and transfer PA in vitro. Crystal structures of the AtSFH5-Sec14 domain in complex with L-α-phosphatidic acid (L-α-PA) and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) revealed that two PA ligands nestled in the central cavity with different configurations, elucidating the specific binding mode of PA to AtSFH5, different from the reported phosphatidylethanolamine (PE)/phosphatidylcholine (PC)/phosphatidylinositol (PI) binding modes. Quantitative lipidomic analysis of chloroplast lipids showed that PA and monogalactosyldiacylglycerol (MGDG), particularly the C18 fatty acids at sn-2 position in MGDG were significantly decreased, indicating a disrupted ER-to-plastid (chloroplast) lipid transfer, under deficiency of AtSFH5 and AtSFH7. Our studies identified the role and elucidated the structural basis of plant SFH proteins in transferring PA between organelles, and suggested a model for ER-chloroplast interorganelle phospholipid transport from inherent ER to chloroplast derived from endosymbiosis of a cyanobacteriumproviding a mechanism involved in the adaptive evolution of cellular plastids.

Cortactin controls bone homeostasis through regulating the differentiation of osteoblasts and osteoclasts.

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis.

Spring Viremia of Carp Virus N Protein Negatively Regulates IFN Induction through Autophagy-Lysosome-Dependent Degradation of STING.

Fish possess a powerful IFN system to defend against aquatic virus infections. Nevertheless, spring viremia of carp virus (SVCV) causes large-scale mortality in common carp and significant economic losses to aquaculture. Therefore, it is necessary to investigate the strategies used by SVCV to escape the IFN response. In this study, we show that the SVCV nucleoprotein (N protein) negatively regulates cellular IFN production by degrading stimulator of IFN genes (STING) via the autophagy-lysosome-dependent pathway. First, overexpression of N protein inhibited the IFN promoter activation induced by polyinosinic-polycytidylic acid and STING. Second, the N protein associated with STING and experiments using a dominant-negative STING mutant demonstrated that the N-terminal transmembrane domains of STING were indispensable for this interaction. Then, the N protein degraded STING in a dose-dependent and autophagy-lysosome-dependent manner. Intriguingly, in the absence of STING, individual N proteins could not elicit host autophagic flow. Furthermore, the autophagy factor Beclin1 was found to interact with the N protein to attenuate N protein-mediated STING degradation after beclin1 knockdown. Finally, the N protein remarkably weakened STING-enhanced cellular antiviral responses. These findings reveal that SVCV uses the host autophagic process to achieve immune escape, thus broadening our understanding of aquatic virus pathogenesis.

Impact of Comorbid Sleep-Disordered Breathing and Atrial Fibrillation on the Long-Term Outcome After Ischemic Stroke.

BACKGROUND: Sleep-disordered breathing (SDB) and atrial fibrillation (AF) are highly prevalent in patients with stroke and are recognized as independent risk factors for stroke. Little is known about the impact of comorbid SDB and AF on long-term outcomes after stroke. METHODS: In this prospective cohort study, 353 patients with acute ischemic stroke or transient ischemic attacks were analyzed. Patients were screened for SDB by respiratory polygraphy during acute hospitalization. Screening for AF was performed using a 7-day ECG up to 3× in the first 6 months. Follow-up visits were scheduled at 1, 3, 12, 24, and 36 months poststroke. Cox regression models adjusted for various factors (age, sex, body mass index, hypertension, diabetes, dyslipidemia, and heart failure) were used to assess the impact of comorbid SDB and AF on subsequent death or cerebro-cardiovascular events. RESULTS: Among 353 patients (299 ischemic stroke and 54 transient ischemic attacks), median age, 67 (interquartile range, 57-74) years with 63% males. Moderate-to-severe SDB (apnea-hypopnea index score, ≥15/h) was present in 118 (33.4%) patients. Among the 56 (15.9%) patients with AF, 28 had comorbid moderate-to-severe SDB and AF. Over 36 months, there were 12 deaths and 67 recurrent cerebro-cardiovascular events. Patients with comorbid moderate-to-severe SDB and AF had a higher risk of subsequent death or cerebro-cardiovascular events compared with those with only moderate-to-severe SDB without AF (hazard ratio, 2.49 [95% CI, 1.18-5.24]) and to those without moderate-to-severe SDB or AF (hazard ratio, 2.25 [95% CI, 1.12-4.50]). However, no significant difference was found between the comorbid moderate-to-severe SDB and AF group and the group with only AF without moderate-to-severe SDB (hazard ratio, 1.64 [95% CI, 0.62-4.36]). CONCLUSIONS: Comorbid moderate-to-severe SDB and AF significantly increase the risk of long-term mortality or recurrent cerebro-cardiovascular events after acute ischemic stroke. Considering both conditions as cumulative and modifiable cerebro-cardiovascular risk factors is of interest for the management of acute stroke. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02559739.

Microsphere-Enhanced Fluorescence-Lightened Solid-Phase Hybridization Assay: The Strategy to Highly Selective Detection of Micro Ribonucleic Acids.

The detection of low-abundance microribonucleic acid (miRNA) frequently adopted nucleic acid sequence-based amplification detection, which was found to have poor selectivity for the nonspecific amplification of template-dependent ligation in enzyme-mediated cascade reactions. Here, a highly selective detection of miRNAs was developed that combined microsphere-enhanced fluorescence (MSEF) and solid-phase base-paired hybridization. The target miRNA could be accurately and quantitatively identified through the solid-phase hybridization assay on the surface of an optical microsphere, while the detected fluorescence signal could be physically amplified by MSEF. Hereinto, the optical microsphere acted as the fluorescence amplifier and whose surface supplied the space to carry out base-paired hybridization to recognize the target miRNA via the immobilized capture DNA sequence. The detected fluorescence signal of the single-base mismatched miRNA-21 sequence was just around 12% of that of the target miRNA-21 sequence in the measurement of model miRNA-21, while the limit of detection of miRNA-21 could be 1.0 fM. The developed detection of miRNA on an optical microsphere was demonstrated to be an excellent physically amplified method to selectively and sensitively detect the target miRNA and magnificently avoid the nonspecific amplification and false-positive results, which is expected to have wide applications in pathematology, pharmacology, clinic diagnosis, and on-site screening fields as well.

WTAP-mediated m6A modification of lncRNA Snhg1 improves myocardial ischemia-reperfusion injury via miR-361-5p/OPA1-dependent mitochondrial fusion.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is caused by reperfusion after ischemic heart disease. LncRNA Snhg1 regulates the progression of various diseases. N6-methyladenosine (m6A) is the frequent RNA modification and plays a critical role in MIRI. However, it is unclear whether lncRNA Snhg1 regulates MIRI progression and whether the lncRNA Snhg1 was modified by m6A methylation. METHODS: Mouse cardiomyocytes HL-1 cells were utilized to construct the hypoxia/reoxygenation (H/R) injury model. HL-1 cell viability was evaluated utilizing CCK-8 method. Cell apoptosis, mitochondrial reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were quantitated utilizing flow cytometry. RNA immunoprecipitation and dual-luciferase reporter assays were applied to measure the m6A methylation and the interactions between lncRNA Snhg1 and targeted miRNA or target miRNAs and its target gene. The I/R mouse model was constructed with adenovirus expressing lncRNA Snhg1. HE and TUNEL staining were used to evaluate myocardial tissue damage and apoptosis. RESULTS: LncRNA Snhg1 was down-regulated after H/R injury, and overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization. Besides, lncRNA Snhg1 could target miR-361-5p, and miR-361-5p targeted OPA1. Overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization though the miR-361-5p/OPA1 axis. Furthermore, WTAP induced lncRNA Snhg1 m6A modification in H/R-stimulated HL-1 cells. Moreover, enforced lncRNA Snhg1 repressed I/R-stimulated myocardial tissue damage and apoptosis and regulated the miR-361-5p and OPA1 levels. CONCLUSION: WTAP-mediated m6A modification of lncRNA Snhg1 regulated MIRI progression through modulating myocardial apoptosis, mitochondrial ROS production, and mitochondrial polarization via miR-361-5p/OPA1 axis, providing the evidence for lncRNA as the prospective target for alleviating MIRI progression.

Small conductance calcium-activated potassium channel contributes to stress induced endothelial dysfunctions.

Patients with Takotsubo syndrome displayed endothelial dysfunction, but underlying mechanisms have not been fully clarified. This study aimed to explore molecular signalling responsible for catecholamine excess induced endothelial dysfunction. Human cardiac microvascular endothelial cells were challenged by epinephrine to mimic catecholamine excess. Patch clamp, FACS, ELISA, PCR, and immunostaining were employed for the study. Epinephrine (Epi) enhanced small conductance calcium-activated potassium channel current (ISK1-3) through activating α1 adrenoceptor. Phenylephrine enhanced edothelin-1 (ET-1) and reactive oxygen species (ROS) production, and the effects involved contribution of ISK1-3. H2O2 enhanced ISK1-3 and ET-1 production. Enhancing ISK1-3 caused a hyperpolarization, which increases ROS and ET-1 production. BAPTA partially reduced phenylephrine-induced enhancement of ET-1 and ROS, suggesting that α1 receptor activation can enhance ROS/ET-1 generation in both calcium-dependent and calcium-independent ways. The study demonstrates that high concentration catecholamine can activate SK1-3 channels through α1 receptor-ROS signalling and increase ET-1 production, facilitating vasoconstriction.

LASS2 suppresses metastasis in multiple cancers by regulating the ferroptosis signalling pathway through interaction with TFRC.

BACKGROUND: As a key enzyme in ceramide synthesis, longevity assurance homologue 2 (LASS2) has been indicated to act as a tumour suppressor in a variety of cancers. Ferroptosis is involved in a variety of tumour processes; however, the role of LASS2 in regulating ferroptosis has yet to be explored. This article explores the potential underlying mechanisms involved. METHODS: Bioinformatics tools and immunohistochemical staining were used to evaluate LASS2 expression, and the results were analysed in relation to overall survival and clinical association in multiple cancers. Coimmunoprecipitation-coupled liquid chromatography-mass spectrometry (co-IP LC-MS) was performed to identify potential LASS2-interacting proteins in thyroid, breast, and liver cancer cell lines. Transcriptomics, proteomics and metabolomics analyses of multiple cancer cell types were performed using MS or LC-MS to further explore the underlying mechanisms involved. Among these tumour cells, the common LASS2 interaction partner transferrin receptor (TFRC) was analysed by protein-protein docking and validated by coimmunoprecipitation western blot, immunofluorescence, and proximity ligation assays. Then, we performed experiments in which tumour cells were treated with Fer-1 or erastin or left untreated, with or without inducing LASS2 overexpression, and assessed the molecular biological and cellular functions by corresponding analyses. RESULTS: Low LASS2 expression is correlated with adverse clinical characteristic and poor prognosis in patients with thyroid cancer, breast cancer or HCC. Multiomics analyses revealed significant changes in the ferroptosis signalling pathway, iron ion transport and iron homeostasis. Our in vitro experiments revealed that LASS2 overexpression regulated ferroptosis status in these tumour cells by affecting iron homeostasis, which in turn inhibited tumour migration, invasion and EMT. In addition, LASS2 overexpression reversed the changes in tumour cell metastasis induced by either Fer-1 or erastin. Mechanistically, LASS2 interacts directly with TFRC to regulate iron homeostasis in these tumour cells. CONCLUSIONS: In summary, our study reveals for the first time that LASS2 can inhibit tumour cell metastasis by interacting with TFRC to regulate iron metabolism and influence ferroptosis status in thyroid, breast, and liver cancer cells, these results suggest potential universal therapeutic targets for the treatment of these cancers.

Elevated liver enzymes in the first trimester are associated with gestational diabetes mellitus: A prospective cohort study.

AIMS: Previous studies have found that a single liver enzyme may predict gestational diabetes mellitus (GDM), but the results have been inconsistent. This study aimed to explore the associations of liver enzymes in early pregnancy with risk of GDM, as well as to independently rank risk factors. METHODS: This prospective cohort study included 1295 women who underwent liver enzyme measurements during early pregnancy and completed GDM assessment in mid-pregnancy. Logistic regression and restricted cubic spline analyses were conducted to assess the relationship between liver enzymes and risk of GDM. Back-propagation artificial neural network was performed to rank independently risk factors of GDM. RESULTS: Women diagnosed with GDM exhibited significantly higher levels of liver enzymes than those without GDM (all p < 0.05). The highest quartile of liver enzymes was associated with higher risk of GDM compared with the lowest quartile, with adjusted odds ratio (ORs) ranging from 2.76 to 8.11 (all p < 0.05). Moreover, the ORs of GDM increased linearly with liver enzymes level (all P for overall association <0.001). Furthermore, Back-propagation artificial neural network identified γ-gamma-glutamyl transferase (GGT) as accounting for the highest proportion in the ranking of GDM risk prediction weights (up to 20.8%). CONCLUSIONS: Single or total elevations of liver enzymes in early pregnancy could predict the GDM occurrence, in which GGT, alkaline Phosphatase, and aspartate aminotransferase were the three most important independent risk factors.

Endothelial BACE1 Impairs Cerebral Small Vessels via Tight Junctions and eNOS.

BACKGROUND: Cerebral small vessel injury, including loss of endothelial tight junctions, endothelial dysfunction, and blood-brain barrier breakdown, is an early and typical pathology for Alzheimer's disease, cerebral amyloid angiopathy, and hypertension-related cerebral small vessel disease. Whether there is a common mechanism contributing to these cerebrovascular alterations remains unclear. Studies have shown an elevation of BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) in cerebral vessels from cerebral amyloid angiopathy or Alzheimer's disease patients, suggesting that vascular BACE1 may involve in cerebral small vessel injury. METHODS: To understand the contribution of vascular BACE1 to cerebrovascular impairments, we combined cellular and molecular techniques, mass spectrometry, immunostaining approaches, and functional testing to elucidate the potential pathological mechanisms. RESULTS: We observe a 3.71-fold increase in BACE1 expression in the cerebral microvessels from patients with hypertension. Importantly, we discover that an endothelial tight junction protein, occludin, is a completely new substrate for endothelial BACE1. BACE1 cleaves occludin with full-length occludin reductions and occludin fragment productions. An excessive cleavage by elevated BACE1 induces membranal accumulation of caveolin-1 and subsequent caveolin-1-mediated endocytosis, resulting in lysosomal degradation of other tight junction proteins. Meanwhile, membranal caveolin-1 increases the binding to eNOS (endothelial nitric oxide synthase), together with raised circulating Aß (ß-amyloid peptides) produced by elevated BACE1, leading to an attenuation of eNOS activity and resultant endothelial dysfunction. Furthermore, the initial endothelial damage provokes chronic reduction of cerebral blood flow, blood-brain barrier leakage, microbleeds, tau hyperphosphorylation, synaptic loss, and cognitive impairment in endothelial-specific BACE1 transgenic mice. Conversely, inhibition of aberrant BACE1 activity ameliorates tight junction loss, endothelial dysfunction, and memory deficits. CONCLUSIONS: Our findings establish a novel and direct relationship between endothelial BACE1 and cerebral small vessel damage, indicating that abnormal elevation of endothelial BACE1 is a new mechanism for cerebral small vessel disease pathogenesis.

Cholecystohepatic shunt pathway reduces secondary bile acid accumulation to enhance natural killer T cell-mediated anti-hepatocellular carcinoma immunity.

BACKGROUND AND AIM: The impact of cholecystectomy, which blocks the cholecystohepatic shunt pathway (CHSP), on the prognosis of patients with hepatocellular carcinoma (HCC) is unclear. Hepatic secondary bile acids (BAs) inhibit natural killer T (NKT) cell-mediated immunity against HCC, and the regulation of homeostasis of hepatic secondary BAs is controlled by the CHSP. However, the influence of CHSP on NKT cell-mediated immunity against HCC remains unclear. METHODS: The clinical data of hospitalized patients undergoing HCC resection were collected. Meanwhile, an in situ HCC mouse model was established, and the CHSP was augmented using oleanolic acid (OA). RESULTS: After 1:1 propensity score matching, Cox regression analysis revealed that cholecystectomy was an independent risk factor for HCC recurrence after hepatectomy (P = 0.027, hazard ratio: 1.599, 95% confidence interval: 1.055-2.422). Experimentally, when OA enhanced CHSP, a significant decrease was observed in the accumulation of secondary BAs in the livers of mice. Additionally, a significant increase was observed in the levels of C-X-C ligand 16 and interferon γ in the serum and tumor tissues. Further, the percentage of C-X-C receptor 6 (+) NKT cells in the tumor tissues increased significantly, and the growth of liver tumors was inhibited. CONCLUSIONS: This clinical study revealed that cholecystectomy promoted the recurrence after radical hepatectomy in patients with HCC. Preserving the normal-functioning gallbladder as much as possible during surgery may be beneficial to the patient's prognosis. Further investigation into the mechanism revealed that CHSP enhanced NKT cell-mediated immunity against HCC by reducing the hepatic accumulation of secondary BAs.

A dynamic computational model of the parallel circuit on the basal ganglia-cortex associated with Parkinson's disease dementia.

The cognitive impairment will gradually appear over time in Parkinson's patients, which is closely related to the basal ganglia-cortex network. This network contains two parallel circuits mediated by putamen and caudate nucleus, respectively. Based on the biophysical mean-field model, we construct a dynamic computational model of the parallel circuit in the basal ganglia-cortex network associated with Parkinson's disease dementia. The simulated results show that the decrease of power ratio in the prefrontal cortex is mainly caused by dopamine depletion in the caudate nucleus and is less related to that in the putamen, which indicates Parkinson's disease dementia may be caused by a lesion of the caudate nucleus rather than putamen. Furthermore, the underlying dynamic mechanism behind the decrease of power ratio is investigated by bifurcation analysis, which demonstrates that the decrease of power ratio is due to the change of brain discharge pattern from the limit cycle mode to the point attractor mode. More importantly, the spatiotemporal course of dopamine depletion in Parkinson's disease patients is well simulated, which states that with the loss of dopaminergic neurons projecting to the striatum, motor dysfunction of Parkinson's disease is first observed, whereas cognitive impairment occurs after a period of onset of motor dysfunction. These results are helpful to understand the pathogenesis of cognitive impairment and provide insights into the treatment of Parkinson's disease dementia.

Ultrasensitive colorimetric detection of Staphylococcus aureus using wheat germ agglutinin and IgY as a dual-recognition strategy.

A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.

Role of membrane fouling layer in microbial fuel cell-membrane bioreactor (MFC-MBR) for controlling sulfamethoxazole and corresponding resistance genes.

Integrated MFC-MBR systems effectively remove antibiotics and control the release of antibiotic resistance genes (ARGs). However, the fouling layers on membranes can potentially act as reservoirs for ARGs. This study aims to elucidate the roles of membrane fouling layers and levels in influencing sulfamethoxazole (SMX) removal and ARGs control within an MFC-MBR system. Our findings demonstrate that low-intensity bioelectricity (400-500 mV) mitigates membrane fouling rates. The membrane fouling layer significantly contributes (39%-47%) to SMX removal compared to the cathode/anode zones. Higher extracellular polymeric substance (EPS) content and a lower protein/polysaccharide (PN/PS) ratio favor SMX removal by the membrane fouling layer. Across different levels of membrane fouling, the PN/PS ratio rather than EPS concentration plays a crucial role in SMX removal efficiency. The MFC-MBR with low fouling achieved superior SMX removal (69.1%) compared to medium (54.3%) and high fouling conditions (46.8%). The presence of ARGs in the membrane fouling layer increases with fouling formation, with intrinsic ARGs prevailing. Dense membrane fouling layers effectively retain ARGs, thereby reducing the risk of extracellular ARGs (eARGs) diffusion in effluents. These results provide insights into controlling ARGs in MFC-MBR systems and underscore the significant role of membrane fouling layers in antibiotics and ARGs removal.

Acid ground nano-realgar processed product inhibits breast cancer by inducing mitophagy via the p53/BNIP3/NIX pathway.

Breast cancer is a highly prevalent malignancy with the first morbidity and the primary reason for female cancer-related deaths worldwide. Acid ground nano-realgar processed product (NRPP) could inhibit breast cancer cell proliferation and induce autophagy in our previous research; however, the underlying mechanisms are still unclear. Therefore, this research aimed to verify whether NRPP induces breast cancer mitophagy and explore the mitophagy-mediated mechanism. Primarily, rhodamine-123 assay and transmission electron microscopy were applied to detect mitochondrial membrane potential (MMP) and ultrastructural changes in the MDA-MB-435S cells, respectively. Mito-Tracker Green/Lyso-Tracker Red staining, western blot, immunofluorescence and RT-PCR were used to explore molecular mechanisms of NRPP-induced mitophagy in vitro. MDA-MB-435S breast cancer xenograft models were established to assess the activity and mechanisms of NRPP in vivo. Our results showed that NRPP decreased MMP and increased autophagosome numbers in MDA-MB-435S cells and activated mitophagy. Furthermore, mitophagy was consolidated because mitochondria and lysosomes colocalized phenomenology were observed, and the expression of LC3II/I and COXIV was upregulated. Additionally, we found the p53/BNIP3/NIX pathway was activated. Finally, NRPP inhibited tumour growth and downregulated the levels of TNF-α, IL-1ß and IL-6. Necrosis, damaged mitochondria and autophagosomes were observed in xenograft tumour cells, and proteins and mRNA levels of LC3, p53, BNIP3 and NIX were increased. Overall, NRPP inhibited MDA-MB-435S cell proliferation and tumour growth by inducing mitophagy via the p53/BNIP3/NIX pathway. Thus, NRPP is a promising candidate for breast cancer treatment.

Analysis of codon usage patterns in 48 Aconitum species.

BACKGROUND: The Aconitum genus is a crucial member of the Ranunculaceae family. There are 350 Aconitum species worldwide, with about 170 species found in China. These species are known for their various pharmacological effects and are commonly used to treat joint pain, cold abdominal pain, and other ailments. Codon usage bias (CUB) analysis contributes to evolutionary relationships and phylogeny. Based on protein-coding sequences (PCGs), we selected 48 species of Aconitum for CUB analysis. RESULTS: The results revealed that Aconitum species had less than 50% GC content. Furthermore, the distribution of GC content was irregular and followed a trend of GC1 > GC2 > GC3, indicating a bias towards A/T bases. The relative synonymous codon usage (RSCU) heat map revealed the presence of conservative codons with slight variations within the genus. The effective number of codons (ENC)-Plot and the parity rule 2 (PR2)-bias plot analysis indicate that natural selection is the primary factor influencing the variation in codon usage. As a result, we screened various optimal codons and found that A/T bases were preferred as the last codon. Furthermore, our Maximum Likelihood (ML) analysis based on PCGs among 48 Aconitum species yielded results consistent with those obtained from complete chloroplast (cp.) genome data. This suggests that analyzing mutation in PCGs is an efficient method for demonstrating the phylogeny of species at the genus level. CONCLUSIONS: The CUB analysis of 48 species of Aconitum was mainly influenced by natural selection. This study reveals the CUB pattern of Aconitum and lays the foundation for future genetic modification and phylogenetic analyses.

ß-aminoisobutyrics acid, a metabolite of BCAA, activates the AMPK/Nrf-2 pathway to prevent ferroptosis and ameliorates lung ischemia-reperfusion injury.

BACKGROUND: Lung ischemia-reperfusion (I/R) injury is a serious clinical problem without effective treatment. Enhancing branched-chain amino acids (BCAA) metabolism can protect against cardiac I/R injury, which may be related to bioactive molecules generated by BCAA metabolites. L-ß-aminoisobutyric acid (L-BAIBA), a metabolite of BCAA, has multi-organ protective effects, but whether it protects against lung I/R injury is unclear. METHODS: To assess the protective effect of L-BAIBA against lung I/R injury, an animal model was generated by clamping the hilum of the left lung, followed by releasing the clamp in C57BL/6 mice. Mice with lung I/R injury were pre-treated or post-treated with L-BAIBA (150 mg/kg/day), given by gavage or intraperitoneal injection. Lung injury was assessed by measuring lung edema and analyzing blood gases. Inflammation was assessed by measuring proinflammatory cytokines in bronchoalveolar lavage fluid (BALF), and neutrophil infiltration of the lung was measured by myeloperoxidase activity. Molecular biological methods, including western blot and immunofluorescence, were used to detect potential signaling mechanisms in A549 and BEAS-2B cells. RESULTS: We found that L-BAIBA can protect the lung from I/R injury by inhibiting ferroptosis, which depends on the up-regulation of the expressions of GPX4 and SLC7A11 in C57BL/6 mice. Additionally, we demonstrated that the Nrf-2 signaling pathway is key to the inhibitory effect of L-BAIBA on ferroptosis in A549 and BEAS-2B cells. L-BAIBA can induce the nuclear translocation of Nrf-2. Interfering with the expression of Nrf-2 eliminated the protective effect of L-BAIBA on ferroptosis. A screening of potential signaling pathways revealed that L-BAIBA can increase the phosphorylation of AMPK, and compound C can block the Nrf-2 nuclear translocation induced by L-BAIBA. The presence of compound C also blocked the protective effects of L-BAIBA on lung I/R injury in C57BL/6 mice. CONCLUSIONS: Our study showed that L-BAIBA protects against lung I/R injury via the AMPK/Nrf-2 signaling pathway, which could be a therapeutic target.

L-BAIBA upregulates the expression of GPX4 and SLC7A11 by activating the AMPK/Nrf-2/GPX4/SLC7A11 signaling pathway, thereby protecting against I/R-induced increase in ROS and ferroptosis in the lung.

Monopolar spindle 1 contributes to tamoxifen resistance in breast cancer through phosphorylation of estrogen receptor α.

PURPOSE: The overexpression of mitotic kinase monopolar spindle 1 (Mps1) has been identified in many tumor types, and targeting Mps1 for tumor therapy has shown great promise in multiple preclinical cancer models. However, the role played by Mps1 in tamoxifen (TAM) resistance in breast cancer has never been reported. METHODS: The sensitivity of breast cancer cells to tamoxifen was analysed in colony formation assays and wound healing assays. Enhanced transactivational activity of estrogen receptor α (ERα) led by Mps1 overexpression was determined by luciferase assays. The interaction between Mps1 and ERα was verified by co-immunoprecipitation and proximity ligation assay. Phosphorylation of ERα by Mps1 was detected by in vitro kinase assay and such phosphorylation process in vivo was proven by co-immunoprecipitation. The potential phosphorylation site(s) of ERα were analyzed by mass spectrometry. RESULTS: Mps1 determines the sensitivity of breast cancer cells to tamoxifen treatment. Mps1 overexpression rendered breast cancer cells more resistant to tamoxifen, while an Mps1 inhibitor or siMps1 oligos enabled cancer cells to overcome tamoxifen resistance. Mechanistically, Mps1 interacted with estrogen receptor α and stimulated its transactivational activity in a kinase activity-dependent manner. Mps1 was critical for ERα phosphorylation at Thr224 amino acid site. Importantly, Mps1 failed to enhance the transactivational activity of the ERα-T224A mutant. CONCLUSION: Mps1 contributes to tamoxifen resistance in breast cancer and is a potential therapeutic that can overcome tamoxifen resistance in breast cancer.

Gastrointestinal symptoms of long COVID-19 related to the ectopic colonization of specific bacteria that move between the upper and lower alimentary tract and alterations in serum metabolites.

BACKGROUND: Since the coronavirus disease 2019 (COVID-19) outbreak, many COVID-19 variants have emerged, causing several waves of pandemics and many infections. Long COVID-19, or long-term sequelae after recovery from COVID-19, has aroused worldwide concern because it reduces patient quality of life after rehabilitation. We aimed to characterize the functional differential profile of the oral and gut microbiomes and serum metabolites in patients with gastrointestinal symptoms associated with long COVID-19. METHODS: We prospectively collected oral, fecal, and serum samples from 983 antibiotic-naïve patients with mild COVID-19 and performed a 3-month follow-up postdischarge. Forty-five fecal and saliva samples, and 25 paired serum samples were collected from patients with gastrointestinal symptoms of long COVID-19 at follow-up and from healthy controls, respectively. Eight fecal and saliva samples were collected without gastrointestinal symptoms of long COVID-19 at follow-up. Shotgun metagenomic sequencing of fecal samples and 2bRAD-M sequencing of saliva samples were performed on these paired samples. Two published COVID-19 gut microbiota cohorts were analyzed for comparison. Paired serum samples were analyzed using widely targeted metabolomics. RESULTS: Mild COVID-19 patients without gastrointestinal symptoms of long COVID-19 showed little difference in the gut and oral microbiota during hospitalization and at follow-up from healthy controls. The baseline and 3-month samples collected from patients with gastrointestinal symptoms associated with long COVID-19 showed significant differences, and ectopic colonization of the oral cavity by gut microbes including 27 common differentially abundant genera in the Proteobacteria phylum, was observed at the 3-month timepoint. Some of these bacteria, including Neisseria, Lautropia, and Agrobacterium, were highly related to differentially expressed serum metabolites with potential toxicity, such as 4-chlorophenylacetic acid, 5-sulfoxymethylfurfural, and estradiol valerate. CONCLUSIONS: Our study characterized the changes in and correlations between the oral and gut microbiomes and serum metabolites in patients with gastrointestinal symptoms associated with long COVID-19. Additionally, our findings reveal that ectopically colonized bacteria from the gut to the oral cavity could exist in long COVID-19 patients with gastrointestinal symptoms, with a strong correlation to some potential harmful metabolites in serum.

Tanshinone IIA (TSIIA) represses the progression of non-small cell lung cancer by the circ_0020123/miR-1299/HMGB3 pathway.

Tanshinone IIA (TSIIA), a multi-pharmaceutical compound, has been demonstrated to have anti-tumor properties. This study explores the potential regulatory mechanism of TSIIA on non-small cell lung cancer (NSCLC) progression. The cytotoxicity of TSIIA was evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and LDH (lactate dehydrogenase) assays. Expression levels of circ_0020123 (hsa_circ_0020123) and microRNA-1299 (miR-1299) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasion, and apoptosis were analyzed by MTT, colony formation, transwell, wound-healing, or flow cytometry assays. The relationship between miR-1299 and circ_0020123 or HMGB3 (high mobility group box 3) was verified by the dual-luciferase reporter and/or RNA immunoprecipitation (RIP) assays. Protein level of HMGB3 was measured by western blotting. The relationship between TSIIA and circ_0020123 was confirmed by xenograft assay. TSIIA reduced xenograft tumor growth in vivo and repressed proliferation, migration, invasion, and facilitated apoptosis of NSCLC cells in vitro. TSIIA reduced circ_0020123 and HMGB3 expression, whereas elevated miR-1299 expression in NSCLC cells. Circ_0020123 knockdown enhanced the repressive influence of TSIIA treatment on the malignancy of NSCLC cells in vitro and in vivo. Circ_0020123 sponged miR-1299 to regulate HMGB3 expression under TSIIA treatment. MiR-1299 inhibitor reversed circ_0020123 knockdown-mediated influence on malignant behaviors of NSCLC cells under TSIIA treatment. HMGB3 elevation offset the suppressive impact of miR-1299 mimic on the malignancy of NSCLC cells under TSIIA treatment. TSIIA curbed NSCLC progression by the circ_0020123/miR-1299/HMGB3 axis, manifesting that the TSIIA/circ_0020123/miR-1299/HMG regulatory network might be a potential treatment strategy for NSCLC.