Dynamic and Label-Free Sensing of Cardiomyocyte Responses to Nanosized Vesicles for Cardiac Oxidative Stress Injury Therapy.

Cardiac oxidative stress is a significant phenotype of myocardial infarction disease, a leading cause of global health threat. There is an urgent need to develop innovative therapies. Nanosized extracellular vesicle (nEV)-based therapy shows promise, yet real-time monitoring of cardiomyocyte responses to nEVs remains a challenge. In this study, a dynamic and label-free cardiomyocyte biosensing system using microelectrode arrays (MEAs) was constructed. Cardiomyocytes were cultured on MEA devices for electrophysiological signal detection and treated with nEVs from E. coli, gardenia, HEK293 cells, and mesenchymal stem cells (MSC), respectively. E. coli-nEVs and gardenia-nEVs induced severe paroxysmal fibrillation, revealing distinct biochemical communication compared to MSC-nEVs. Principal component analysis identified variations and correlations between nEV types. MSC-nEVs enhanced recovery without inducing arrhythmias in a H2O2-induced oxidative stress injury model. This study establishes a fundamental platform for assessing biochemical communication between nEVs and cardiomyocytes, offering new avenues for understanding nEVs' functions in the cardiovascular system.

Capilliposide B inhibits the migration of prostate cancer by inducing autophagy through the ROS/AMPK/mTOR pathway.

Capilliposide B (CPS-B), a novel oleanane triterpenoid saponin derived from Lysimachia capillipes Hemsl, is a potent anticancer agent. However, its anticancer mechanism remains elusive. In the present study, we demonstrated the potent anti-tumor activity and molecular mechanism of CPS-B both in vitro and in vivo. Proteomic analysis using isobaric tags for relative and absolute quantitation techniques suggested that CPS-B modulated autophagy in prostate cancer (PC). Moreover, Western blotting showed that both autophagy and epithelial-mesenchymal transition occurred place after CPS-B treatment in vivo, which was also proven in PC-3 cancer cells. We deduced that CPS-B inhibited migration by inducing autophagy. We examined the accumulation of reactive oxygen species (ROS) in cells, and in downstream pathways, LKB1 and AMPK were activated while mTOR was inhibited. Transwell experiment results showed that CPS-B inhibited the metastasis of PC-3 cells and that this effect was significantly attenuated after pretreatment with chloroquine, indicating that CPS-B inhibited metastasis via autophagy induction. Altogether, these data suggest that CPS-B is a potential therapeutic agent for cancer treatment that acts by inhibiting migration through the ROS/AMPK/mTOR signaling pathway.

Idelalisib inhibits experimental proliferative vitroretinopathy.

Proliferative vitreoretinopathy (PVR) is a fibrotic eye disease that develops after rhegmatogenous retinal detachment surgery and open-globe traumatic injury. Idelalisib is a specific inhibitor of phosphoinositide 3-kinase (PI3K) δ. While PI3Kδ is primarily expressed in leukocytes, its expression is also considerably high in retinal pigment epithelial (RPE) cells, which play a crucial part in the PVR pathogenesis. Herein we show that GeoMx Digital Spatial Profiling uncovered strong expression of fibronectin in RPE cells within epiretinal membranes from patients with PVR, and that idelalisib (10 µM) inhibited Akt activation, fibronectin expression and collagen gel contraction induced by transforming growth factor (TGF)-ß2 in human RPE cells. Furthermore, we discovered that idelalisib at a vitreal concentration of 10 µM, a non-toxic dose to the retina, prevented experimental PVR induced by intravitreally injected RPE cells in rabbits assessed by experienced ophthalmologists using an indirect ophthalmoscope plus a + 30 D fundus lens, electroretinography, optical coherence tomography and histological analysis. These data suggested idelalisib could be harnessed for preventing patients from PVR.

Guangsangon E triggers mitochondria dysfunction and mitophagy in triple-negative breast cancer and leads to non-apoptotic cell death.

Guangsangon E (GSE) is a natural product separated from Morus alba L. It has been reported to treat lung cancer through autophagy. However, whether GSE is effective in repressing triple-negative breast cancer (TNBC) cells is yet to be elucidated. In the present study, GSE inhibited cell growth of MDA-MB-231, MDA-MB-453, and MDA-MB-468 cells. Moreover, GSE induced mitochondrial dysfunction, including membrane potential loss, mitochondria fission, and reactive oxygen species accumulation, and finally led to mitophagy-related non-apoptotic cell death. In the xenograft tumor nude mice, GSE treatment significantly reduced the size and weight of MDA-MB-231 tumors. The tumor inhibition rates of GSE treatment were 49.68% (low-dose) and 48.73% (high-dose). In summary, GSE is a potential anticancer drug available for treating TNBC with apoptosis resistance.

Chalcomoracin prevents vitreous-induced activation of AKT and migration of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells are the major cell type in the epi- or sub-retinal membranes in the pathogenesis of proliferative vitreoretinopathy (PVR), which is a blinding fibrotic eye disease and still short of effective medicine. The purpose of this study is to demonstrate whether Chalocomoracin (CMR), a novel purified compound from fungus-infected mulberry leaves, is able to inhibit vitreous-induced signalling events and cellular responses intrinsic to PVR. Our studies have revealed that the CMR IC50 for ARPE-19 cells is 35.5 µmol/L at 72 hours, and that 5 µmol/L CMR inhibits vitreous-induced Akt activation and p53 suppression; in addition, we have discovered that this chemical effectively blocks vitreous-stimulated proliferation, migration and contraction of ARPE-19 cells, suggesting that CMR is a promising PVR prophylactic.

A novel Diels-Alder adduct of mulberry leaves exerts anticancer effect through autophagy-mediated cell death.

Guangsangon E (GSE) is a novel Diels-Alder adduct isolated from leaves of Morus alba L, a traditional Chinese medicine widely applied in respiratory diseases. It is reported that GSE has cytotoxic effect on cancer cells. In our research, we investigated its anticancer effect on respiratory cancer and revealed that GSE induces autophagy and apoptosis in lung and nasopharyngeal cancer cells. We first observed that GSE inhibits cell proliferation and induces apoptosis in A549 and CNE1 cells. Meanwhile, the upregulation of autophagosome marker LC3 and increased formation of GFP-LC3 puncta demonstrates the induction of autophagy in GSE-treated cells. Moreover, GSE increases the autophagy flux by enhancing lysosomal activity and the fusion of autophagosomes and lysosomes. Next, we investigated that endoplasmic reticulum (ER) stress is involved in autophagy induction by GSE. GSE activates the ER stress through reactive oxygen species (ROS) accumulation, which can be blocked by ROS scavenger NAC. Finally, inhibition of autophagy attenuates GSE-caused cell death, termed as "autophagy-mediated cell death." Taken together, we revealed the molecular mechanism of GSE against respiratory cancer, which demonstrates great potential of GSE in the treatment of representative cancer.

Phosphoinositide 3-kinase δ inactivation prevents vitreous-induced activation of AKT/MDM2/p53 and migration of retinal pigment epithelial cells.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kß, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR.

Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes.

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

Blockade of MDM2 with inactive Cas9 prevents epithelial to mesenchymal transition in retinal pigment epithelial cells.

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-ß2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-ß2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-ß2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-ß2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-ß2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-ß2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-ß2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-ß2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells.

Mouse double minute (MDM)2 single nucleotide polymorphism (SNP) 309G allele in the second promoter of MDM2 enhances vitreous-induced expression of Mdm2 and degradation of the tumor suppressor protein p53. This MDM2SNP309G contributes to certain cancer development and experimental proliferative vitreoretinopathy. The goal of this study is to discover a novel strategy to only block vitreous-induced expression of Mdm2 for preventing vitreous-induced cell proliferation and survival and thus find a potential novel strategy to treat proliferation-related diseases. We created two mutations (D10A and H840A) in Streptococcus pyogenes (Sp)Cas9 within the nuclease domains (RuvC1 and HNH, respectively) to render this SpCas9 nuclease dead named as dCas9 in a lentiCRISPR v2 vector. Then an MDM2-sgRNA targeting the second promoter of human MDM2 gene was cloned into this vector for producing lentivirus to infect human retinal pigment epithelial (RPE) cells with, which carry a heterozygous genotype of MDM2SNP309 T/G. lacZ-sgRNA was used as a control. As a result, we discovered that vitreous from experimental rabbits induced a 1.9 ±â€¯0.2 fold increase in Mdm2 and a 2.0 ±â€¯0.2 fold decrease in p53 in the RPE cells with dCas9/lacZ-sgRNA compared to those with dCas9/MDM2-sgRNA, suggesting that dCas9 under the guidance of the MDM2-sgRNA prevented RV-stimulated increase in Mdm2. In addition, we found that the rabbit vitreous significantly enhanced cell proliferation (1.5 ±â€¯0.2 fold), survival against apoptosis (2.2 ±â€¯0.2 fold), migration (10 ±â€¯1.5%) and contraction (112.7 ±â€¯14.1 mm2) of the cells with dCas9/lacZ-sgRNA compared with those with dCas9/MDM2-sgRNA. These results indicated that application of the dCas9 targeted to the P2 of MDM2 is a potential therapeutic approach to diseases due to the P2-driven aberrant expression of Mdm2 - such as proliferative vitreoretinopathy.

Elevating intracellular action potential recording in cardiomyocytes: A precision-enhanced and biosafe single-pulse electroporation system.

Action potentials play a pivotal role in diverse cardiovascular physiological mechanisms. A comprehensive understanding of these intricate mechanisms necessitates a high-fidelity intracellular electrophysiological investigative approach. The amalgamation of micro-/nano-electrode arrays and electroporation confers substantial advantages in terms of high-resolution intracellular recording capabilities. Nonetheless, electroporation systems typically lack precise control, and commonly employed electroporation modes, involving tailored sequences, may escalate cellular damage and perturbation of normal physiological functions due to the multiple or higher-intensity electrical pulses. In this study, we developed an innovative electrophysiological biosensing system customized to facilitate precise single-pulse electroporation. This advancement serves to achieve optimal and uninterrupted intracellular action potential recording within cardiomyocytes. The refinement of the single-pulse electroporation technique is realized through the integration of the electroporation and assessment biosensing system, thereby ensuring a consistent and reliable means of achieving stable intracellular access. Our investigation has unveiled that the optimized single-pulse electroporation technique not only maintains robust biosafety standards but also enables the continuous capture of intracellular electrophysiological signals across an expansive three-day period. The universality of this biosensing system, adaptable to various micro/nano devices, furnishes real-time analysis and feedback concerning electroporation efficacy, guaranteeing the sustained, secure, and high-fidelity acquisition of intracellular data, thereby propelling the field of cardiovascular electrophysiological research.

A smart tablet-phone-based high-performance pancreatic cancer cell biosensing system for drug screening.

Exploring more efficient pancreatic cancer drug screening platforms is of significant importance for accelerating the drug development process. In this study, we developed a high-sensitivity bioluminescence system based on smartphones and smart tablets, and constructed a pancreatic cancer drug screening platform (PCDSP) by combining the pancreatic cancer cell sensing model (PCCSM) on the multiwell plates (MTP). A smart tablet was used as the light source and a smartphone as the colorimetric sensing device. The smartphone dynamically controls the color and brightness displayed on the smart tablet to achieve lower LOD and wider detection ranges. We constructed PCCSM for 24 h, 48 h, and 72 h , and performed colorimetric experiments using both PCDSP and a commercial plate reader (CPR). The results showed that the PCDSP had a lower LOD than that of CPR. Moreover, PCDSP even exhibited a lower LOD for 24 h PCCSM testing compared to CPR for 48 h PCCSM testing, effectively shortening the drug evaluation process. Additionally, the PCDSP offers higher portability and efficiency compared with CPR, making it a promising platform for efficient pancreatic cancer drug screening.

Deciphering the metabolic profile and anti-colorectal cancer mechanism of Capilliposide A using ultra performance liquid chromatography mass spectrometry combined with non-targeted metabolomics studies.

Colorectal cancer is a highly prevalent malignancy that threatens human health worldwide. Despite the availability of chemotherapy as a primary treatment option, individuals with CRC undergoing frequent chemotherapy are susceptible to developing drug resistance, which can result in poor treatment outcomes. Consequently, there is an urgent need to discover new bioactive compounds for the treatment of CRC. Capilliposide A is a triterpenoid saponin that is extracted from Lysimachia capillipes Hemsl. Although it has been reported that LC-A exhibits good bioactivity, its metabolic profile and potential mechanism underlying its anti-CRC effects remain unknown. In this study, the metabolic products of LC-A in rat plasma, feces, and urine were identified using an LC-MS platform. In addition, LC-MS-based metabolomics was employed to investigate the mechanism of LC-A against CRC. The results showed that LC-A significantly inhibited CRC cell proliferation, attenuated tumor growth, and alleviated metabolic abnormalities in CRC-bearing mice. Furthermore, the levels of p-cresol sulfate and phenylacetylglycine in CRC model plasma decreased, with an increment in sphingosine 1-phosphate, D-tryptophan, and L-2-aminoadipic acid. These metabolite levels can be reversed by LC-A treatment. These metabolite alterations were related to the sphingolipid and amino acid metabolic pathways, demonstrating that LC-A anti-CRC effects were regulated through the modulation of underlying metabolism. Additionally, seven metabolites of LC-A were characterized in rat feces, plasma, and urine. This study offers a scientific foundation for elucidating the metabolism of LC-A and its treatment of colorectal cancer.

Biomimetic integrated gustatory and olfactory sensing array based on HL-1 cardiomyocyte facilitating drug screening for tachycardia treatment.

The ectopic co-expression of taste and olfactory receptors in cardiomyocytes provides not only possibilities for the construction of biomimetic gustatory and olfactory sensors but also promising novel therapeutic targets for tachycardia treatment. Here, bitter taste and olfactory receptors endogenously expressed in HL-1 cells were verified by RT-PCR and immunofluorescence staining. Then HL-1 cardiomyocyte-based integrated gustatory and olfactory sensing array coupling with the microelectrode array (MEA) was first constructed for drugs screening and evaluation for tachycardia treatment. The MEA sensor detected the extracellular field potentials and reflected the systolic-diastolic properties of cardiomyocytes in real time in a label-free and non-invasive way. The in vitro tachycardia model was constructed using isoproterenol as the stimulator. The proposed sensing array facilitated potential drug screening for tachycardia treatment, such as salicin, artemisinin, xanthotoxin, and azelaic acid which all activated specific receptors on HL-1 cells. IC50 values for four potential drugs were calculated to be 0.0036 µM, 309.8 µM, 14.68 µM, and 0.102 µM, respectively. Visualization analysis with heatmaps and PCA cluster showed that different taste and odorous drugs could be easily distinguished. The mean inter-class Euclidean distance between different bitter drugs was 1.681, which was smaller than the distance between bitter and odorous drugs of 2.764. And the inter-class distance was significantly higher than the mean intra-class Euclidean distance of 1.172. In summary, this study not only indicates a new path for constructing novel integrated gustatory and olfactory sensors but also provides a powerful tool for the quantitative evaluation of potential drugs for tachycardia treatment.

NFκB-Mediated Expression of Phosphoinositide 3-Kinase δ Is Critical for Mesenchymal Transition in Retinal Pigment Epithelial Cells.

Epithelial mesenchymal transition (EMT) plays a vital role in a variety of human diseases including proliferative vitreoretinopathy (PVR), in which retinal pigment epithelial (RPE) cells play a key part. Transcriptomic analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was up-regulated in human RPE cells upon treatment with transforming growth factor (TGF)-ß2, a multifunctional cytokine associated with clinical PVR. Stimulation of human RPE cells with TGF-ß2 induced expression of p110δ (the catalytic subunit of PI3Kδ) and activation of NFκB/p65. CRISPR-Cas9-mediated depletion of p110δ or NFκB/p65 suppressed TGF-ß2-induced fibronectin expression and activation of Akt as well as migration of these cells. Intriguingly, abrogating expression of NFκB/p65 also blocked TGF-ß2-induced expression of p110δ, and luciferase reporter assay indicated that TGF-ß2 induced NFκB/p65 binding to the promoter of the PIK3CD that encodes p110δ. These data reveal that NFκB/p65-mediated expression of PI3Kδ is essential in human RPE cells for TGF-ß2-induced EMT, uncovering hindrance of TGF-ß2-induced expression of p110δ as a novel approach to inhibit PVR.

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis.

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.

Mitochondrial proteomic analysis reveals the regulation of energy metabolism and reactive oxygen species production in Clematis terniflora DC. leaves under high-level UV-B radiation followed by dark treatment.

Clematis terniflora DC. is an important medicinal plant from the family Ranunculaceae. A previous study has shown that active ingredients in C. terniflora, such as flavonoids and coumarins, are increased under ultraviolet B radiation (UV-B) and dark treatment and that the numbers of genes related to the tricarboxylic acid cycle and mitochondrial electron transport chain (mETC) are changed. To uncover the mechanism of the response to UV-B radiation and dark treatment in C. terniflora, mitochondrial proteomics was performed. The results showed that proteins related to photorespiration, mitochondrial membrane permeability, the tricarboxylic acid cycle, and the mETC mainly showed differential expression profiles. Moreover, the increase in alternative oxidase indicated that another oxygen-consuming respiratory pathway in plant mitochondria was induced to minimize mitochondrial reactive oxygen species production. These results suggested that respiration and mitochondrial membrane permeability were deeply influenced to avoid energy consumption and maintain energy balance under UV-B radiation and dark treatment in C. terniflora leaf mitochondria. Furthermore, oxidative phosphorylation was able to regulate intracellular oxygen balance to resist oxidative stress. This study improves understanding of the function of mitochondria in response to UV-B radiation and dark treatment in C. terniflora. SIGNIFICANCE: C. terniflora was an important traditional Chinese medicine for anti-inflammatory. Previous study showed that the contents of coumarins which were the main active ingredient in C. terniflora were induced by UV-B radiation and dark treatment. In the present study, to uncover the regulatory mechanism of metabolic changes in C. terniflora, mitochondrial proteomics analysis of leaves was performed. The results showed that photorespiration and oxidative phosphorylation pathways were influenced under UV-B radiation and dark treatment. Mitochondria in C. terniflora leaf played a crucial role in energy mechanism and regulation of cellular oxidation-reduction to maintain cell homeostasis under UV-B radiation followed with dark treatment.

Comparative Proteomic and Metabolomic Analyses of Plasma Reveal the Novel Biomarker Panels for Thyroid Dysfunction.

BACKGROUND: Thyroid dysfunction, including hypothyroidism (THO) and hyperthyroidism (THE), commonly arise from pathological processes in the thyroid gland. The current diagnosis of thyroid dysfunction varies because of the age and sex of the patients. The aim of this study was to explore novel candidate biomarker panels for hypothyroidism and hyperthyroidism screening with mass spectrometry and bioinformatics. METHODS: Plasma samples were collected from 15 THE patients, 9 THO patients, and 15 healthy controls. Data Independent Acquisition(DIA)-based proteomic and untargeted metabolomic analyses were performed to identify novel biomarker panels for THO and THE patients. Finally, three candidate biomarkers were verified by ELISA in 34 samples. RESULTS: A total of 2738 proteins and 6103 metabolites were identified, and 173 proteins and 2487 metabolites were found to be differentially expressed among the THE, THO and control groups. The results of the ensemble feature selection, K-means clustering and least absolute shrinkage and selection operator (LASSO) regression model showed that two proteins (C4-A and C3/C5 convertase) combined with two metabolites (L-arginine and L-proline), and proteins (APOL1 and ITIH4) combined with metabolites (cortisol, and cortisone) identified by plasma proteomics and metabolomics could help distinguish THO and THE patients from healthy controls, respectively. CONCLUSIONS: This study identified and verified two pairs of biomarker panels that can be used to distinguish THE and THO patients regardless of age and sex. Consequently, our findings represent a comprehensive analysis of thyroid dysfunction plasma, which is significant for clinical diagnosis.

Combined ultraviolet and darkness regulation of medicinal metabolites in Mahonia bealei revealed by proteomics and metabolomics.

Roots of Mahonia bealei have been used as traditional Chinese medicine with antibacterial, antioxidant and anti-inflammatory properties due to its high alkaloid content. Previously, we reported that alkaloid and flavonoid contents in the M. bealei leaves could be increased by the combined ultraviolet B and dark treatment (UV+D). To explore the underlying metabolic pathways and networks, proteomic and metabolomic analyses of the M. bealei leaves were conducted. Proteins related to tricarboxylic acid cycle, transport and signaling varied greatly under the UV + D. Among them, calmodulin involved in calcium signaling and ATP-binding cassette transporter involved in transport of berberine were increased. Significantly changed metabolites were overrepresented in phenylalanine metabolism, nitrogen metabolism, phenylpropanoid, flavonoid and alkaloid biosynthesis. In addition, the levels of salicylic acid and gibberellin decreased in the UV group and increased in the UV + D group. These results indicate that multi-hormone crosstalk may regulate the biosynthesis of flavonoids and alkaloids to alleviate oxidative stress caused by the UV + D treatment. Furthermore, protoberberine alkaloids may be induced through calcium signaling crosstalk with reaction oxygen species and transported to leaves. SIGNIFICANCE: Mahonia bealei root and stem, not leaf, were used as traditional medicine for a long history because of the high contents of active components. In the present study, UV-B combined with dark treatments induced the production of alkaloids and flavonoids in the M. bealei leaf, especially protoberberine alkaloids such as berberine. Multi-omics analyses indicated that multi-hormone crosstalk, enhanced tricarboxylic acid cycle and active calcium signaling were involved. The study informs a strategy for utilization of the leaves, and improves understanding of the functions of secondary metabolites in M. bealei.