Neural mechanisms of sequential dependence in time perception: the impact of prior task and memory processing.

Our perception and decision-making are susceptible to prior context. Such sequential dependence has been extensively studied in the visual domain, but less is known about its impact on time perception. Moreover, there are ongoing debates about whether these sequential biases occur at the perceptual stage or during subsequent post-perceptual processing. Using functional magnetic resonance imaging, we investigated neural mechanisms underlying temporal sequential dependence and the role of action in time judgments across trials. Participants performed a timing task where they had to remember the duration of green coherent motion and were cued to either actively reproduce its duration or simply view it passively. We found that sequential biases in time perception were only evident when the preceding task involved active duration reproduction. Merely encoding a prior duration without reproduction failed to induce such biases. Neurally, we observed activation in networks associated with timing, such as striato-thalamo-cortical circuits, and performance monitoring networks, particularly when a "Response" trial was anticipated. Importantly, the hippocampus showed sensitivity to these sequential biases, and its activation negatively correlated with the individual's sequential bias following active reproduction trials. These findings highlight the significant role of memory networks in shaping time-related sequential biases at the post-perceptual stages.

Subtype-Specific Association of Mitochondrial DNA Copy Number With Poststroke/TIA Outcomes in 10†241 Patients in China.

BACKGROUND: Mitochondrial DNA copy number (mtDNA-CN) is associated with the severity and mortality in patients with stroke, but the associations in different stroke subtypes remain unexplored. METHODS: We conducted an observational prospective cohort analysis on patients with ischemic stroke or transient ischemic attack enrolled in the Third China National Stroke Registry. We applied logistic models to assess the association of mtDNA-CN with functional outcome (modified Rankin Scale score, 3-6 versus 0-2) and Cox proportional hazard models to assess the association with stroke recurrence (treating mortality as a competing risk) and mortality during a 12-month follow-up, adjusting for sex, age, physical activity, National Institutes of Health Stroke Scale at admission, history of stroke and peripheral artery disease, small artery occlusion, and interleukin-6. Subgroup analyses stratified by age and stroke subtypes were conducted. RESULTS: The Third China National Stroke Registry enrolled 15 166 patients, of which 10 241 with whole-genome sequencing data were retained (mean age, 62.2 [SD, 11.2] years; 68.8% men). The associations between mtDNA-CN and poststroke/transient ischemic attack outcomes were specific to patients aged ≤65 years, with lower mtDNA-CN significantly associated with stroke recurrence in 12 months (subdistribution hazard ratio, 1.15 per SD lower mtDNA-CN [95% CI, 1.04-1.27]; P=5.2×10-3) and higher all-cause mortality in 3 months (hazard ratio, 2.19 [95% CI, 1.41-3.39]; P=5.0×10-4). Across subtypes, the associations of mtDNA-CN with stroke recurrence were specific to stroke of undetermined cause (subdistribution hazard ratio, 1.28 [95% CI, 1.11-1.48]; P=6.6×10-4). In particular, lower mtDNA-CN was associated with poorer functional outcomes in stroke of undetermined cause patients diagnosed with embolic stroke of undetermined source (odds ratio, 1.53 [95% CI, 1.20-1.94]; P=5.4×10-4), which remained significant after excluding patients with recurrent stroke (odds ratio, 1.49 [95% CI, 1.14-1.94]; P=3.0×10-3). CONCLUSIONS: Lower mtDNA-CN is associated with higher stroke recurrence rate and all-cause mortality, as well as poorer functional outcome at follow-up, among stroke of undetermined cause, embolic stroke of undetermined source, and younger patients.

Enhancing ventrolateral prefrontal cortex activation mitigates social pain and modifies subsequent social attitudes: Insights from TMS and fMRI.

Social pain, a multifaceted emotional response triggered by interpersonal rejection or criticism, profoundly impacts mental well-being and social interactions. While prior research has implicated the right ventrolateral prefrontal cortex (rVLPFC) in mitigating social pain, the precise neural mechanisms and downstream effects on subsequent social attitudes remain elusive. This study employed transcranial magnetic stimulation (TMS) integrated with fMRI recordings during a social pain task to elucidate these aspects. Eighty participants underwent either active TMS targeting the rVLPFC (n = 41) or control stimulation at the vertex (n = 39). Our results revealed that TMS-induced rVLPFC facilitation significantly reduced self-reported social pain, confirming the causal role of the rVLPFC in social pain relief. Functional connectivity analyses demonstrated enhanced interactions between the rVLPFC and the dorsolateral prefrontal cortex, emphasizing the collaborative engagement of prefrontal regions in emotion regulation. Significantly, we observed that negative social feedback led to negative social attitudes, whereas rVLPFC activation countered this detrimental effect, showcasing the potential of the rVLPFC as a protective buffer against adverse social interactions. Moreover, our study uncovered the impact role of the hippocampus in subsequent social attitudes, a relationship particularly pronounced during excitatory TMS over the rVLPFC. These findings offer promising avenues for improving mental health within the intricate dynamics of social interactions. By advancing our comprehension of the neural mechanisms underlying social pain relief, this research introduces novel intervention strategies for individuals grappling with social distress. Empowering individuals to modulate rVLPFC activation may facilitate reshaping social attitudes and successful reintegration into communal life.

Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behaviors in mice.

BACKGROUND: Major depressive disorder (MDD) is a common but severe psychiatric illness characterized by depressive mood and diminished interest. Both nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 1 (NLRP1) inflammasome and autophagy have been reported to implicate in the pathological processes of depression. However, the mechanistic interplay between NLRP1 inflammasome, autophagy, and depression is still poorly known. METHODS: Animal model of depression was established by chronic social defeat stress (CSDS). Depressive-like behaviors were determined by social interaction test (SIT), sucrose preference test (SPT), open field test (OFT), forced swim test (FST), and tail-suspension test (TST). The protein expression levels of NLRP1 inflammasome complexes, pro-inflammatory cytokines, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K)/PI3K, phosphorylated-AKT (p-AKT)/AKT, phosphorylated-mechanistic target of rapamycin (p-mTOR)/mTOR, brain-derived neurotrophic factor (BDNF), phosphorylated-tyrosine kinase receptor B (p-TrkB)/TrkB, Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl2) and cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3) were examined by western blotting. The mRNA expression levels of pro-inflammatory cytokines were tested by quantitative real-time PCR. The interaction between proteins was detected by immunofluorescence and coimmunoprecipitation. Neuronal injury was assessed by Nissl staining. The autophagosomes were visualized by transmission electron microscopy. Nlrp1a knockdown was performed using an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. RESULTS: CSDS exposure caused a bidirectional change in hippocampal autophagy function, which was activated in the initial period but impaired at the later stage. In addition, CSDS exposure increased the expression levels of hippocampal NLRP1 inflammasome complexes, pro-inflammatory cytokines, p-PI3K, p-AKT and p-mTOR in a time-dependent manner. Interestingly, NLRP1 is immunoprecipitated with mTOR but not PI3K/AKT and CSDS exposure facilitated the immunoprecipitation between them. Hippocampal Nlrp1a knockdown inhibited the activity of PI3K/AKT/mTOR signaling, rescued the impaired autophagy and ameliorated depressive-like behavior induced by CSDS. In addition, rapamycin, an autophagy inducer, abolished NLRP1 inflammasome-driven inflammatory reactions, alleviated depressive-like behavior and exerted a neuroprotective effect. CONCLUSIONS: Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behavior in mice and the regulation of autophagy could be a valuable therapeutic strategy for the management of depression.

Non-O blood types are associated with a greater risk of large artery atherosclerosis stroke and dysregulation of cholesterol metabolism: an observational study.

BACKGROUND: Previous research on ABO blood types and stroke has been controversial, predominantly suggesting heightened risk of stroke in non-O blood types. Nonetheless, investigations into the correlation and underlying mechanisms between ABO blood groups and stroke subtypes, especially within Chinese cohorts, remain limited. METHODS: The ABO blood types of 9,542 ischaemic stroke (IS) patients were inferred using two ABO gene loci (c.261G > del; c.802G > A). The healthy population was derived from the 1000 Genomes Project. Patients were classified by the causative classification system (CCS). Volcano plot and gene ontology (GO) analysis were employed to explore protein differential expression among blood types. Additionally, HT29 and SW480 cell lines with downregulated ABO expression were generated to evaluate its impact on cholesterol uptake and efflux. RESULTS: A greater proportion of stroke patients had non-O blood types (70.46%) than did healthy individuals (61.54%). Notable differences in blood type distributions were observed among stroke subtypes, with non-O blood type patients mainly classified as having large artery atherosclerosis (LAA). Clinical baseline characteristics, such as the low-density lipoprotein cholesterol level, activated partial thromboplastin time and thrombin time, varied significantly among blood types. A volcano plot revealed 17 upregulated and 42 downregulated proteins in the O blood type. GO term analysis indicated that downregulated proteins were primarily associated with lipid metabolism pathways. In vitro experiments revealed that reducing ABO gene expression decreased cholesterol uptake and increased cholesterol efflux. CONCLUSIONS: This study revealed that the non-O blood type increased the risk of LAA stroke through cholesterol metabolism.

A Multiple Targeting Nanoprobe for Identifying Cancer Metastatic Sites Based on Detection of Various mRNAs in Circulating Tumor Cells.

Identification of cancer metastatic sites is of importance for adjusting therapeutic interventions and treatment choice. However, identifying the location of metastatic lesions with easy accessibility and high safety is challenging. Here we demonstrate that cancer metastatic sites can be accurately detected by a triple targeting nanoprobe. Through coencapsulating molecular beacons probing a cancer biomarker (CXCR4 mRNA), a lung metastatic biomarker (CTSC mRNA), and a bone metastatic biomarker (JAG1 mRNA), the nanoprobe decorated by SYL3C conjugated hyaluronic acid and ICAM-1 specific aptamer conjugated hyaluronic acid can target diverse phenotyped circulating tumor cells (CTCs) during epithelial-mesenchymal and mesenchymal-epithelial transitions in whole blood for sensitive probing. The detection of CTCs from cancer patients shows that the nanoprobe can provide accurate information to distinguish different cancer metastasis statuses including nonmetastasis, lung metastasis, and bone metastasis. This study proposes an efficient screening tool for identifying the location of distant metastatic lesions via facile blood biopsy.

Development and validation of a nomogram to predict the risk of constipation after lumbar interbody fusion surgery.

INTRODUCTION: To understand the incidence of postoperative constipation and the risk factors of constipation in patients with lumbar interbody fusion, we constructed and verified the constipation risk prediction model, so as to provide reference for the prevention and treatment of postoperative constipation. METHODS: The data of patients undergoing lumbar interbody fusion in our hospital were retrospectively analyzed from December 2021 to December 2022. According to postoperative constipation, the patients were divided into constipation group and non-constipation group. Univariate logistic regression analysis and multivariate logistic regression analysis were used to determine independent risk factors for postoperative constipation. Based on independent risk factors, a nomogram was developed to predict the risk of constipation after lumbar interbody fusion. The prediction performance was assessed using receiver operating characteristic curve (ROC), calibration curve and decision curve analysis (DCA). Finally, bootstrapping method for internal validation was further evaluated the nomogram. RESULTS: A total of 282 patients participated in the study. 176 patients (62.41%) after lumbar interbody occurred constipation, and 106 patients were asymptomatic. Multivariate regression analysis showed independent risk factors, including the use of calcium channel blockers, polypharmacy, postoperative bed time, and constipation history. Multivariate regression analysis was used to establish the model. The C-index of the nomogram was 0.827 (95% CI 0.779-0.875), and the C-index of interval bootstrapping validation was 0.813 (95% CI 0.765-0.861), and the area under the AUC was 0.800. The nomogram showed good discrimination ability. CONCLUSIONS: The use of calcium channel blockers, polypharmacy, postoperative bed time, and history of constipation are independent risk factors for postoperative constipation in patients undergoing lumbar interbody fusion. The constructed risk prediction model has good discriminative ability.

A pipeline for sample tagging of whole genome bisulfite sequencing data using genotypes of whole genome sequencing.

BACKGROUND: In large-scale high-throughput sequencing projects and biobank construction, sample tagging is essential to prevent sample mix-ups. Despite the availability of fingerprint panels for DNA data, little research has been conducted on sample tagging of whole genome bisulfite sequencing (WGBS) data. This study aims to construct a pipeline and identify applicable fingerprint panels to address this problem. RESULTS: Using autosome-wide A/T polymorphic single nucleotide variants (SNVs) obtained from whole genome sequencing (WGS) and WGBS of individuals from the Third China National Stroke Registry, we designed a fingerprint panel and constructed an optimized pipeline for tagging WGBS data. This pipeline used Bis-SNP to call genotypes from the WGBS data, and optimized genotype comparison by eliminating wildtype homozygous and missing genotypes, and retaining variants with identical genomic coordinates and reference/alternative alleles. WGS-based and WGBS-based genotypes called from identical or different samples were extensively compared using hap.py. In the first batch of 94 samples, the genotype consistency rates were between 71.01%-84.23% and 51.43%-60.50% for the matched and mismatched WGS and WGBS data using the autosome-wide A/T polymorphic SNV panel. This capability to tag WGBS data was validated among the second batch of 240 samples, with genotype consistency rates ranging from 70.61%-84.65% to 49.58%-61.42% for the matched and mismatched data, respectively. We also determined that the number of genetic variants required to correctly tag WGBS data was on the order of thousands through testing six fingerprint panels with different orders for the number of variants. Additionally, we affirmed this result with two self-designed panels of 1351 and 1278 SNVs, respectively. Furthermore, this study confirmed that using the number of genetic variants with identical coordinates and ref/alt alleles, or identical genotypes could not correctly tag WGBS data. CONCLUSION: This study proposed an optimized pipeline, applicable fingerprint panels, and a lower boundary for the number of fingerprint genetic variants needed for correct sample tagging of WGBS data, which are valuable for tagging WGBS data and integrating multi-omics data for biobanks.

The ubiquitin-binding protein MdRAD23D1 mediates drought response by regulating degradation of the proline-rich protein MdPRP6 in apple (Malus domestica).

RAD23 (RADIATION SENSITIVE23) proteins are a group of UBL-UBA (ubiquitin-like-ubiquitin-associated) proteins that shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Drought stress is a major environmental constraint that limits plant growth and production, but whether RAD23 proteins are involved in this process is unclear. Here, we demonstrated that a shuttle protein, MdRAD23D1, mediated drought response in apple plants (Malus domestica). MdRAD23D1 levels increased under drought stress, and its suppression resulted in decreased stress tolerance in apple plants. Through in vitro and in vivo assays, we demonstrated that MdRAD23D1 interacted with a proline-rich protein MdPRP6, resulting in the degradation of MdPRP6 by the 26S proteasome. And MdRAD23D1 accelerated the degradation of MdPRP6 under drought stress. Suppression of MdPRP6 resulted in enhanced drought tolerance in apple plants, mainly because the free proline accumulation is changed. And the free proline is also involved in MdRAD23D1-mediated drought response. Taken together, these findings demonstrated that MdRAD23D1 and MdPRP6 oppositely regulated drought response. MdRAD23D1 levels increased under drought, accelerating the degradation of MdPRP6. MdPRP6 negatively regulated drought response, probably by regulating proline accumulation. Thus, "MdRAD23D1-MdPRP6" conferred drought stress tolerance in apple plants.

Variants within the LPL gene confer susceptility to diabetic kidney disease and rapid decline in kidney function in Chinese patients with type 2 diabetes.

AIM: To examine the association between lipoprotein lipase (LPL) polymorphisms and susceptibility to diabetic kidney disease (DKD) and early renal function decline in Chinese patients with type 2 diabetes (T2D). METHODS: The association of eight LPL single nucleotide polymorphisms (SNPs) with DKD was analysed in 2793 patients with T2D from the third China National Stroke Registry. DKD was defined as either an urine albumin-to-creatinine ratio (UACR) of 30 mg/g or higher at baseline and 3 months, or an estimated glomerular filtration rate (eGFR) of less than 60 mL/min/1.73 m2 at baseline and 3 months. Rapid decline in kidney function (RDKF) was defined as a reduction in the eGFR of 3 mL/min/1.73 m2 or greater per year. Logistic regression models were used to evaluate the association of LPL SNP and DKD with an additive model. RESULTS: The SNPs rs285 C>T (OR = 1.40, P = .0154), rs328 C>G (OR = 2.24, P = .0104) and rs3208305 A>T (OR = 1.85, P = .0015) were identified to be significantly associated with DKD defined by eGFR. Among 1241 participants with follow-up data, 441 (35.5%) showed RDKF over a mean follow-up period of 1 year, and the rs285 C allele was associated with higher odds of RDKF (OR = 1.31, 95% CI 1.04-1.66; P = .025) after adjustment for multiple variables. CONCLUSIONS: These results suggest that LPL-related SNPs are new candidate factors for conferring susceptibility to DKD and may promote rapid loss of renal function in Chinese patients with T2D.

Self-assembly synthesis of hollow phosphorus-doped graphitic carbon nitride microboxes for the photodegradation of organic pollutants.

The rational design of photocatalysts with efficiency and stability is highly desirable but remains challenging. Here, we report a supramolecular self-assembly strategy to construct hollow phosphorus-doped g-C3N4 microboxes (PCNMs). Considering the effects of multiple parameters on the structure and activity of samples, the orthogonal design is innovatively introduced to optimize technology parameters for screening high-performance g-C3N4. Under visible light irradiation (λ ≥ 420 nm), rhodamine B (RhB, 4 mg L-1) is completely degraded in just 80 seconds in the presence of the optimal PCNM. The kinetic rate constant of RhB degradation with the PCNM is 3.4633 min-1, demonstrating unprecedented activity that is about 112 times higher than that of bulk g-C3N4 (0.0309 min-1) synthesized by direct polycondensation of melamine. Additionally, the optimal PCNM also shows enhanced degradation efficiency for tetracycline. The outstanding properties are primarily attributed to the hollow architecture, high specific surface area, and phosphorus doping. This work advances the design of photocatalysts correlating various factors, opening an avenue for optimizing photocatalytic synthesis and activity.

The gut microbiome and intestinal failure-associated liver disease.

Intestinal failure-associated liver disease (IFALD) is a common hepatobiliary complication resulting from long-term parenteral nutrition (PN) in patients with intestinal failure. The spectrum of IFALD ranges from cholestasis, steatosis, portal fibrosis, to cirrhosis. Development of IFALD is a multifactorial process, in which gut dysbiosis plays a critical role in its initiation and progression in conjunction with increased intestinal permeability, activation of hepatic immune responses, and administration of lipid emulsion. Gut microbiota manipulation including pre/probiotics, fecal microbiota transplantation, and antibiotics has been studied in IFALD with varying success. In this review, we summarize current knowledge on the taxonomic and functional changes of gut microbiota in preclinical and clinical studies of IFALD. We also review the function of microbial metabolites and associated signalings in the context of IFALD. By providing microbiota-targeted interventions aiming to optimize PN-induced liver injury, our review provides perspectives for future basic and translational investigations in the field.

Comparison of the efficacy and safety of different surgical procedures for patients with hemorrhoids: a network meta-analysis.

PURPOSE: This study used a network meta-analysis to evaluate the efficacy and safety of different surgical approaches in patients with hemorrhoids. METHODS: PubMed, Embase, Web of science, and Cochrane Library were searched for randomized controlled trials on patients with hemorrhoids treated by different surgical procedures. The search was conducted until January 15, 2023. Two investigators independently screened the resulting literature, extracted information, evaluated the risk of bias of the included studies, and performed a network meta-analysis. RESULT: A total of 23 randomized controlled studies were included and involved 3573 patients and 10 interventions, namely L (Ligasure), M-M (Milligan-Morgan), F (Ferguson), H (Harmonic), OH (open Harmonic), CH (closed Harmonic), PPH (procedure for prolapse and hemorrhoids), TST (tissue selecting technique), T-S (TST STARE+; tissue selection therapy stapled transanal rectal resection plus), and STARR (stapled transanal rectal resection). Network meta-analysis results showed that L has the shortest mean operating time and STARR has the longest mean operating time, F and H have the longest length of hospitalization and T-S has the shortest length of hospitalization, PPH has the most intraoperative blood loss and L has the least intraoperative blood loss, TST has the shortest time to first defecation and M-M has the longest time to first defecation, STARR had the least recurrence and PPH had the most recurrence, PPH had the least anal stenosis and L had the most anal stenosis, and F had the least postoperative pain after 24 h and PPH had the most postoperative pain after 24 h. CONCLUSION: Current evidence suggests that L is best at reducing mean operative time and intraoperative bleeding, T-S is best at reducing mean length of stay, TST has the shortest time to first defecation, STARR is best at reducing recurrence rates, PPH is best at reducing postoperative anal stricture, and F is best at reducing postoperative pain after 24 h.

Recent Progress in Gas Sensors Based on P3HT Polymer Field-Effect Transistors.

In recent decades, the rapid development of the global economy has led to a substantial increase in energy consumption, subsequently resulting in the emission of a significant quantity of toxic gases into the environment. So far, gas sensors based on polymer field-effect transistors (PFETs), a highly practical and cost-efficient strategy, have garnered considerable attention, primarily attributed to their inherent advantages of offering a plethora of material choices, robust flexibility, and cost-effectiveness. Notably, the development of functional organic semiconductors (OSCs), such as poly(3-hexylthiophene-2,5-diyl) (P3HT), has been the subject of extensive scholarly investigation in recent years due to its widespread availability and remarkable sensing characteristics. This paper provides an exhaustive overview encompassing the production, functionalization strategies, and practical applications of gas sensors incorporating P3HT as the OSC layer. The exceptional sensing attributes and wide-ranging utility of P3HT position it as a promising candidate for improving PFET-based gas sensors.

Probiotic Spore-Based Oral Drug Delivery System for Enhancing Pancreatic Cancer Chemotherapy by Gut-Pancreas-Axis-Guided Delivery.

The chemotherapeutic effectiveness of pancreatic ductal adenocarcinoma (PDAC) is severely hampered by insufficient intratumoral delivery of antitumor drugs. Here, we demonstrate that enhanced pancreatic cancer chemotherapy can be achieved by probiotic spore-based oral drug delivery system via gut-pancreas axis translocation. Clostridium butyricum spores resistant to harsh external stress are extracted as drug carriers, which are further covalently conjugated with gemcitabine-loaded mesoporous silicon nanoparticles (MGEM). The spore-based oral drug delivery system (SPORE-MGEM) migrates upstream into pancreatic tumors from the gut, which increases intratumoral drug accumulation by ∼3-fold compared with MGEM. In two orthotopic PDAC mice models, tumor growth is markedly suppressed by SPORE-MGEM without obvious side effects. Leveraging the biological contact of the gut-pancreas axis, this probiotic spore-based oral drug delivery system reveals a new avenue for enhancing PDAC chemotherapy.

Detection of mRNAs of Ribosomal Protein L15 and E-Cadherin in Living Circulating Tumor Cells at Single Cell Resolution To Study Tumor Heterogeneity.

To study the heterogeneity of circulating tumor cells (CTCs) is of crucial importance to analyze cancer progression and metastasis. However, in situ detection of highly heterogeneous CTCs in peripheral blood still faces an elusive challenge. Here, we show direct detection of two metastasis-related mRNAs of diverse CTCs in whole blood by a triple-targeting nanoprobe. In the nanoprobe, two kinds of molecular beacons, MB1 to detect RPL15 mRNA and MB2 to detect E-cadherin (E-cad) mRNA, are loaded in a highly efficient delivery vector decorated with EpCAM-targeted SYL3C, EGFR-targeted CL4, and CD44-targeted hyaluronic acid chains to specifically deliver MB1/MB2 into epithelial, mesenchymal, and stem CTCs in unprocessed peripheral blood. The numbers of RPL15+ and E-cad+ CTCs are positively correlated with the metastasis stages of cancer patients. This study provides an effective strategy to realize direct observation on diverse metastasis-related genes in living CTCs with different phenotypes to provide accurate information on cancer heterogeneity and metastasis.

In Situ Detection of Nanotoxicity in Living Cells Based on Multiple miRNAs Probed by a Peptide Functionalized Nanoprobe.

The potential toxicity of nanoparticles, especially for clinically applicable ones, has become a critical concern. Technologies that can in situ-evaluate the toxicity of nanoparticles with high sensitivity are urgently needed. In this study, a facile strategy was developed for sensitive detection on the nanotoxicity of nanoparticles with low toxicity or a low dose. A functional nanoprobe loaded with molecular beacons was constructed to realize in situ evaluation of the nanotoxicity through probing multiple miRNAs in nanoparticle-exposed living cells. Being composed of protamine complexed with molecular beacons for miRNA detection and decorated by TAT and KALA peptides, the dual-peptide functionalized nanoprobe can efficiently deliver molecular beacons into living cells to realize the real-time monitoring of early biomarkers (miR-21 and miR-221) to evaluate nanotoxicity. Using mesoporous silica nanoparticles (MSNs) with different surface modifications as typical representatives of low toxic nanoparticles, we demonstrate that our nanoprobe can sensitively detect miRNA changes in cells under diverse exposure conditions, that is, MSN-NH2 exhibits the strongest capability to upregulate miR-21 and miR-221, and the upregulation is exposure dose- and time-dependent. Our approach is much more sensitive as compared with conventional methods to study cytotoxicity such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell morphology observation, and reactive oxygen species (ROS) assay. This study paves a path for effective and facile nanotoxicity evaluation and provides insights into the biological impacts of MSNs.

Precise Detection on Cell-Cell Fusion by a Facile Molecular Beacon-Based Method.

Cell-cell fusion studies provide an experimental platform for evaluating disease progression and investigating cell infection. However, to realize sensitive and quantitative detection on cell-cell fusion is still a challenge. Herein, we report a facile molecular beacon (MB)-based method for precise detection on cell-cell fusion. By transfection of the spike protein (S protein) and enhanced green fluorescent protein (EGFP) in HEK 293 cells, the virus-mimicking fusogenic effector cells 293-S-EGFP cells were constructed to interact with target cells. Before mixing the effector cells with the target cells, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in 293-S-EGFP cells was silenced, and the MB for GAPDH mRNA detection was delivered into the GAPDH silenced 293-S-EGFP cells. Once cell-cell fusion occurred, MB migrated from the GAPDH silenced effector cells to the target cells and hybridized with GAPDH mRNA in the target cells to induce fluorescence emission. The cell-cell fusion can be easily visualized and quantitated by fluorescence microscopy and flow cytometry. The fluorescence intensity is strongly dependent on the number of fused target cells. This MB-based method can easily identify the differences in the cell fusions for various target cells with different angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) expression levels, resulting in dramatically different fluorescence intensities in fused target cells. Our study provides a convenient and efficient quantitative detection approach to study cell-cell fusion.

Knockdown of estrogen-related receptor α inhibits valve interstitial cell calcification in vitro by regulating heme oxygenase 1.

Calcific aortic valve disease (CAVD) is the most common valvular heart disease in adults. The cellular mechanisms of CAVD are still unknown, but accumulating evidence has revealed that osteogenic differentiation of human valve interstitial cells (hVICs) plays an important role in CAVD. Thus, we aimed to investigate the function of estrogen-related receptor α (ERRα) in the osteogenic differentiation of hVICs. We found that the level of ERRα was significantly increased in CAVD samples compared to normal controls. In addition, ERRα was significantly upregulated during hVIC osteogenic differentiation in vitro. Gain- and loss-of-function experiments were performed to identify the function of ERRα in hVIC calcification in vitro. Inhibition of endogenous ERRα attenuated hVIC calcification, whereas overexpression of ERRα in hVICs promoted this process. RNA sequencing results suggested that heme oxygenase-1 (Hmox1) was a downstream target of ERRα, which was further confirmed by western blotting. Additionally, we also found that downregulation of Hmox1 by shHmox1 efficiently reversed the inhibition of calcification induced by ERRα shRNA in hVICs. ChIP-qPCR and luciferase assays indicated that Hmox1 was negatively regulated by ERRα. We found that overexpression of Hmox1 or its substrates significantly inhibited hVIC calcification in vitro. In conclusion, we found that knockdown of ERRα can inhibit hVIC calcification through upregulating Hmox1 and that ERRα and Hmox1 are potential targets for the treatment of CAVD.

A Unique Chemo-photodynamic Antitumor Approach to Suppress Hypoxia via Ultrathin Graphitic Carbon Nitride Nanosheets Supported a Platinum(IV) Prodrug.

Tumor hypoxia severely restrains the efficiency of irreversible O2-consumption photodynamic therapy. The deep hypoxia induced by photodynamic therapy can promote the level of hypoxia inducible factor 1α that participates in many tumor processes and eventually lead to poor therapeutic outcomes. Herein, a chemo-photodynamic antitumor strategy based on ultrathin graphitic carbon nitride nanosheets loaded with a hypoxia-targeting platinum(IV) prodrug is reported. Under low-intensity visible light irradiation, such integrated nanosheets effectively generate reactive oxygen species together with DNA binding platinum species to achieve enhanced antiproliferation efficacy by downregulating HIF-1α under hypoxic conditions.