Real-time in situ fluorescence imaging of telomerase and miR378 in living cells using a two-color DNA tetrahedron nanoprobe combined with molecular beacons.

A biosensor that can detect biomarkers accurately, quickly, and conveniently is important for the diagnosis of various diseases. However, most of the existing detection methods require sample extraction, which makes it difficult to detect and image intracellular molecules or to detect two different types of biomarkers simultaneously. In this study, we constructed a DNA tetrahedral nanoprobe (DTP) capable of detecting both miR378 and telomerase, both of which are tumor markers. In the presence of miR378, FAM on the molecular beacon of DTP fluoresced via Förster resonance energy transfer (FRET), and the limit of detection was 476 pM with excellent specificity. When present, telomerase binds to telomerase substrate (TS) primers, extending the repeat sequence (TTAGGG)n to trigger Cy3 fluorescence. A strong linear relationship existed between the fluorescence intensity of Cy3 and the number of HeLa cells. The limit of detection was 800 HeLa cells. In addition, DTP was less cytotoxic to and biocompatible with HeLa cells and fluoresced only in cancer cells, which can help to sensitively distinguish between normal and cancer cells. In conclusion, DTP can simultaneously detect the content of miR378 and activity of telomerase and realize intracellular imaging, which has broad application prospects in early cancer diagnosis and treatment.

Exerkines and osteoarthritis.

Osteoarthritis (OA) is the most prevalent chronic joint disease, with physical exercise being a widely endorsed strategy in its management guidelines. Exerkines, defined as cytokines secreted in response to acute and chronic exercise, function through endocrine, paracrine, and/or autocrine pathways. Various tissue-specific exerkines, encompassing exercise-induced myokines (muscle), cardiokines (heart), and adipokines (adipose tissue), have been linked to exercise therapy in OA. Exerkines are derived from these kines, but unlike them, only kines regulated by exercise can be called exerkines. Some of these exerkines serve a therapeutic role in OA, such as irisin, metrnl, lactate, secreted frizzled-related protein (SFRP), neuregulin, and adiponectin. While others may exacerbate the condition, such as IL-6, IL-7, IL-15, IL-33, myostatin, fractalkine, follistatin-like 1 (FSTL1), visfatin, activin A, migration inhibitory factor (MIF), apelin and growth differentiation factor (GDF)-15. They exerts anti-/pro-apoptosis/pyroptosis/inflammation, chondrogenic differentiation and cell senescence effect in chondrocyte, synoviocyte and mesenchymal stem cell. The modulation of adipokine effects on diverse cell types within the intra-articular joint emerges as a promising avenue for future OA interventions. This paper reviews recent findings that underscore the significant role of tissue-specific exerkines in OA, delving into the underlying cellular and molecular mechanisms involved.

Pioglitazone protects PC12 cells against oxidative stress injury: An in vitro study of its antiapoptotic effects via the PPARγ pathway.

To the best of our knowledge, the role of peroxisome proliferator-activated receptor γ (PPARγ) in oxidative stress-induced PC12 cell damage is unknown. Using a PC12 cell model with H2O2 treatment, the present study investigated the expression levels of apoptosis-related genes and neuronal apoptosis after oxidative stress injury. The present study further investigated the protective effect and mechanism of pioglitazone, a PPARγ agonist. PC12 cells treated with H2O2 were used as a model of oxidative stress injury. An MTT assay and flow cytometry were used to detect the effect of H2O2 on PC12 cell viability and the protective effect of pioglitazone. A TUNEL assay was used to detect neuronal apoptosis. The expression levels of PPARγ, Bax, Bcl-2 and caspase-3 were examined by reverse transcription-quantitative PCR and western blotting. H2O2 reduced PC12 cell viability in a dose- and time-dependent manner. H2O2 significantly upregulated the protein expression levels of Bax and the cleaved caspase-3/caspase-3 ratio (P<0.01), decreased the protein expression levels of Bcl-2 (P<0.01), and increased the apoptosis rate of PC12 cells. Pioglitazone significantly reduced the protein expression levels of Bax and the cleaved caspase-3/caspase-3 ratio (P<0.01), increased the expression levels of Bcl-2 (P<0.01), decreased the Bax/Bcl-2 expression ratio (P<0.01) and increased the viability of H2O2-damaged PC12 cells in a dose-dependent manner. Treatment with the PPARγ antagonist GW9662 or PPARγ small interfering RNA counteracted the protective effect of pioglitazone on PC12 cells to different extents (P<0.01). Therefore, the present study reported the role of PPARγ in protecting PC12 cells against oxidative stress injury, which may lead to novel therapeutic approaches for neurodegenerative diseases.

Predicting the risk of distant and local recurrence for patients with ocular adnexal extranodal marginal zone lymphoma: a matched case-control study.

BACKGROUND/AIMS: Extranodal marginal zone lymphoma of ocular adnexa (OA-EMZL) is the most frequent type of ocular adnexal lymphomas, with a high rate of disease recurrence. Precise patient stratification based on disease recurrence is understudied. This study aims to identify risk factors of distant recurrence (DR) and local recurrence (LR) to construct a prognostic model optimising rapid decision of therapeutic strategies. METHODS: A total of 104 patients diagnosed with OA-EMZL between January 2011 and February 2020 were enrolled. Propensity score matching was performed for DR and LR groups. A nomogram was generated using a multivariate Cox proportional hazards model. RESULTS: After matching, different independent risk factors of DR and LR were identified. Monocyte percentage (p=0.015) and M category >0 (p=0.043) were significant independent risk factors of DR. Epiphora (p<0.001) was the significant independent risk factor of LR. Three factors (monocyte percentage, M category >0, age >60) were integrated into the nomogram to predict the risk of DR. It had a relatively better discriminative ability for distant recurrence-free survival (C-index: 3-year, 0.784; 6-year, 0.801) than IPI score (C-index: 3-year, 0.663; 6-year, 0.673) in the cohort of all patients. CONCLUSION: Our analyses suggested DR and LR as two distinct prognostic events, and additionally identified novel risk factors of them. The nomogram may serve as a practical tool for the prognostic estimation and rapid decision of therapeutic strategies for patients with OA-EMZL.

Proteotranscriptomics of ocular adnexal B-cell lymphoma reveals an oncogenic role of alternative splicing and identifies a diagnostic marker.

BACKGROUND: Ocular adnexal B-cell lymphoma (OABL) is a rare subtype of non-Hodgkin lymphoma. The molecular characteristics of OABL remain poorly understood. We performed an integrated study to investigate the proteotranscriptome landscape and identify novel molecular characteristics and biomarkers of OABL. METHODS: Integrated quantitative proteome and transcriptome were performed on 40 OABL 12 idiopathic orbital inflammation, 6 reactive lymphoid hyperplasia, and 13 aesthetic orbital plastic surgery specimens. Complete clinicopathologic and prognostic data of the patients were recorded. RESULTS: We identified high global protein-mRNA concordance as a novel characteristic of OABL. High concordance was related to OABL recurrence. By integrated expression profile, motif enrichment and trend analysis, we found that alternative splicing is inflammation-independently dysregulated in OABL. After portraying the aberrant alternative splicing event landscape, we demonstrated the oncogenic role of ADAR, a core splicing regulator that regulates the splicing of Rho GTPase and cell cycle members. We found that ADAR regulates cell proliferation and Rho GTPase inhibitor sensitivity of lymphoma. We identified DNAJC9 as a potential biomarker for OABL in proteomic analyses. Immunohistochemistry and immunofluorescent staining showed the nuclear staining of DNAJC9 was significantly higher in extranodal marginal zone lymphomas compared with inflammation specimens. CONCLUSIONS: These results provide an integrated gene expression profiling and demonstrate that high global protein-mRNA concordance is a prognosis-related molecular characteristic of OABL. We portray the alternative splicing events landscape of OABL, and reveal the oncogenic role of ADAR. We identified strong nuclear staining of DNAJC9 as a promising pathology diagnostic biomarker for extranodal marginal zone lymphomas.

Ammonium removal using a calcined natural zeolite modified with sodium nitrate.

Ammonium is one of the key factors responsible for the eutrophication of water bodies. The purpose of this study was to remove ammonium from water using a natural zeolite (NZ) modified with sodium nitrate (NaNO3) by impregnation and calcination. The ability of the NZ to remove ammonium from water was determined by single calcination; however, its efficiency was significantly enhanced by impregnation with a NaNO3 solution. Zeolite modified with 3.00 M NaNO3 and calcination at 673 K yielded the best ammonium removal efficiency, which was 39.88 % higher than the NZ alone. The zeolites that were regenerated over six times maintained a removal rate of 79.35-84.79 % by mixing 25.0 mg of the NZ into 50 mL of a 5.0 mg/L ammonium solution. The improved performance of the modified zeolite (qm, 16.96 mg/g) was mainly attributed to its relatively elevated mesopore volumes and higher ion-exchange capacity that results from nitrate decomposition, oxygen release, and sodium-ion exchange. The adsorption kinetics and isotherms are best described by the pseudo-first-order (PFO) and Freundlich model, respectively, and the process was endothermic. The effects of other factors, including coexisting ions, pH, and dosage, on ammonium adsorption were also determined.

Distinct effects of mutations on biophysical properties of human prion protein monomers and oligomers.

Prion diseases are a group of fatal neurodegenerative illnesses, resulting from the conformational conversion of the cellular prion protein (PrPC) into a misfolded form (PrPSc). The formation of neurotoxic soluble prion protein oligomer (PrPO) is regarded as a key step in the development of prion diseases. About 10%-15% of human prion diseases are caused by mutations in the prion protein gene; however, the underlying molecular mechanisms remain unclear. In the present work, we compared the biophysical properties of wild-type (WT) human prion protein 91-231 (WT HuPrP91-231) and its disease-associated variants (P105L, D178N, V203I, and Q212P) using several biophysical techniques. In comparison with WT HuPrPC, the Q212P and D178N variants possessed greatly increased conversion propensities of PrPC into PrPO, while the V203I variant had dramatically decreased conversion propensity. The P105L variant displayed a similar conversion propensity to WT HuPrPC Guanidine hydrochloride-induced unfolding experiments ranked the thermodynamic stabilities of these proteins as Q212P < D178N < WT ≈ P105L < V203I. It was thus suggested that the conversion propensities of the prion proteins are closely associated with their thermodynamic stabilities. Furthermore, structural comparison illustrated that Q212P, D178N, and V203I variants underwent larger structural changes compared with WT HuPrPC, while the P105L variant adopted a similar structure to the WT HuPrPC The mutation-induced structural perturbations might change the thermodynamic stabilities of the HuPrPC variants, and correspondingly alter the conversion propensities for these prion proteins. Our results extend the mechanistic understanding of prion pathogenesis, and lay the basis for the prevention and treatment of prion diseases.

Unique Properties of the Rabbit Prion Protein Oligomer.

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders infecting both humans and animals. Recent works have demonstrated that the soluble prion protein oligomer (PrPO), the intermediate of the conformational transformation from the host-derived cellular form (PrPC) to the disease-associated Scrapie form (PrPSc), exerts the major neurotoxicity in vitro and in vivo. Rabbits show strong resistance to TSEs, the underlying mechanism is unclear to date. It is expected that the relative TSEs-resistance of rabbits is closely associated with the unique properties of rabbit prion protein oligomer which remain to be addressed in detail. In the present work, we prepared rabbit prion protein oligomer (recRaPrPO) and human prion protein oligomer (recHuPrPO) under varied conditions, analyzed the effects of pH, NaCl concentration and incubation temperature on the oligomerization, and compared the properties of recRaPrPO and recHuPrPO. We found that several factors facilitated the formation of prion protein oligomers, including low pH, high NaCl concentration, high incubation temperature and low conformational stability of monomeric prion protein. RecRaPrPO was formed more slowly than recHuPrPO at physiological-like conditions (< 57°C, < 150 mM NaCl). Furthermore, recRaPrPO possessed higher susceptibility to proteinase K and lower cytotoxicity in vitro than recHuPrPO. These unique properties of recRaPrPO might substantially contribute to the TSEs-resistance of rabbits. Our work sheds light on the oligomerization of prion proteins and is of benefit to mechanistic understanding of TSEs-resistance of rabbits.

Distinct effects of Cu2+-binding on oligomerization of human and rabbit prion proteins.

The cellular prion protein (PrP(C)) is a kind of cell-surface Cu(2+)-binding glycoprotein. The oligomerization of PrP(C) is highly related to transmissible spongiform encephalopathies (TSEs). Cu(2+) plays a vital role in the oligomerization of PrP(C), and participates in the pathogenic process of TSE diseases. It is expected that Cu(2+)-binding has different effects on the oligomerization of TSE-sensitive human PrP(C) (HuPrP(C)) and TSE-resistant rabbit PrP(C) (RaPrP(C)). However, the details of the distinct effects remain unclear. In the present study, we measured the interactions of Cu(2+) with HuPrP(C) (91-230) and RaPrP(C) (91-228) by isothermal titration calorimetry, and compared the effects of Cu(2+)-binding on the oligomerization of both PrPs. The measured dissociation constants (Kd) of Cu(2+) were 11.1 ± 2.1 µM for HuPrP(C) and 21.1 ± 3.1 µM for RaPrP(C). Cu(2+)-binding promoted the oligomerization of HuPrP(C) more significantly than that of RaPrP(C). The far-ultraviolet circular dichroism spectroscopy experiments showed that Cu(2+)-binding induced more significant secondary structure change and increased more ß-sheet content for HuPrP(C) compared with RaPrP(C). Moreover, the urea-induced unfolding transition experiments indicated that Cu(2+)-binding decreased the conformational stability of HuPrP(C) more distinctly than that of RaPrP(C). These results suggest that RaPrP(C) possesses a low susceptibility to Cu(2+), potentially weakening the risk of Cu(2+)-induced TSE diseases. Our work sheds light on the Cu(2+)-promoted oligomerization of PrP(C), and may be helpful for further understanding the TSE-resistance of rabbits.