DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA.

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL-1, 1.5 copies⋅µL-1, and 0.17 copies⋅µL-1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

Optimization and mechanisms of proteolytic enzyme immobilization onto large-pore mesoporous silica nanoparticles: Enhanced tumor penetration.

Immobilization of proteolytic enzymes onto nanocarriers is effective to improve drug diffusion in tumors through degrading the dense extracellular matrix (ECM). Herein, immobilization and release behaviors of hyaluronidase, bromelain, and collagenase (Coll) on mesoporous silica nanoparticles (MSNs) were explored. A series of cationic MSNs (CMSNs) with large and adjustable pore sizes were synthesized, and investigated together with two anionic MSNs of different pore sizes. CMSNs4.0 exhibited the highest enzyme loading capacity for hyaluronidase and bromelain, and CMSNs4.5 was the best for Coll. High electrostatic interaction, matched pore size, and large pore volume and surface area favor the immobilization. Changes of the enzyme conformations and surface charges with pH, existence of a space around the immobilized enzymes, and the depth of the pore structures, affect the release ratio and tunability. The optimal CMSNs-enzyme complexes exhibited deep and homogeneous penetration into pancreatic tumors, a tumor model with the densest ECM, with CMSNs4.5-Coll as the best. Upon loading with doxorubicin (DOX), the CMSNs-enzyme complexes induced high anti-tumor efficiencies. Conceivably, the DOX/CMSNs4.5-NH2-Coll nanodrug exhibited the most effective tumor therapy, with a tumor growth inhibition ratio of 86.1 %. The study provides excellent nanocarrier-enzyme complexes, and offers instructive theories for enhanced tumor penetration and therapy.

O2-Generation-Enhanced Responsive Starvation/Photothermal Synergistic Tumor Therapy Based on the AuNRs@MnO2@SiO2 Nanocarrier and Thermosensitive Biomimetic Camouflaging.

Cancer starvation/photothermal combined tumor therapy (CST/PTT) has attracted great interest attributed to their mutual compensation and synergistically enhanced effect. However, the very low O2 supply in the tumor microenvironment (TME) greatly limits the CST efficiency of glucose oxidase (GOx). Additionally, the easy degradation in blood circulation and significant off-target effects are big challenges for clinical applications of the GOx-based CST. In this study, a drug delivery system (DDS) with specific tumor-targeted GOx delivery, near-infrared (NIR) light and TME responsive O2 generation, NIR-responsive glucose consumption, high GOx loading, and efficient NIR photothermia was developed. Positively charged AuNRs@MnO2@SiO2 nanoparticles (named AMS+ NPs) were synthesized. GOx was covalently loaded with a high loading ratio of 36.0%. Finally, a thermosensitive biomimetic hybrid membrane composed of a thermosensitive lipid (TSL) membrane, red blood cell membrane (RBCM), and 4T1 cancer cell membrane (CCM) was coated on the NPs through a double-layer strategy. The AMS+-G@TSL@[RBC-CC-TSL]M NPs consumed 32.7 times glucose at 50 °C as that at 37 °C and generated 4.9 times O2 upon NIR laser irradiation. The thermosensitive biomimetic NPs showed an efficient targeting capability to the homotypic 4T1 cancer cells/tumors accompanied by good biocompatibility, macrophage evading capability, high cancer cell cytotoxicity, and excellent antitumor efficacy. The tumor growth inhibition ratio with NIR laser irradiation reached 92.8%. The AMS+-GOx@TSL@[RBC-CC-TSL]M NPs provide a smart, efficient, safe, PTT/CST combined DDS for highly efficient tumor therapy.

pH-Sensitive Biomimetic Nanosystem Based on Large-Pore Mesoporous Silica Nanoparticles with High Hyaluronidase Loading for Tumor Deep Penetration.

Loading hyaluronidase (Hyal) in a nanocarrier is a potent strategy to degrade the tumor extracellular matrix for tumor deep penetration and enhanced tumor therapy. Herein, a pH-sensitive biomimicking nanosystem with high Hyal loading, effective tumor targeting, and controllable release is constructed. Specifically, cationic mesoporous silica nanoparticles (CMSNs) with large pores 13.52 nm in diameter were synthesized in a one-pot manner by adding N-[3-trimethoxysilylpropyl]-N,N,N-trimethylammonium to a reversed microemulsion reaction system. The Hyal loading rate was as high as 19.47% owing to matched pore size and the cationic surface charge. Subsequently, a pH-sensitive biomimetic hybrid membrane (pHH) composed of pH-sensitive liposome (pHL), red blood cell membrane, and pancreatic cancer cell membrane was camouflaged on the pHL-coated and doxorubicin/Hyal-loaded CMSNs (shortened as DHCM). The DHCM@pHL@pHH is stable at neutral pH while it releases the payloads smoothly in the tumor acidic microenvironment. Consequently, it can escape from macrophage clearance, be specifically taken up by pancreatic cancer cells, and efficiently accumulate at the tumor site. More importantly, it can penetrate deeply in pancreatic tumors with a tumor growth inhibition ratio of 80.46%. The nanosystem is biocompatible and has potential for clinical transformation, and the nanocarrier is promisingly applicable as a platform for encapsulation of various macromolecules for smart and tumor-targeted delivery.

Aptamer-based technology for gastric cancer theranostics.

Gastric cancer is one of the most common causes of cancer death worldwide. This cancer exhibits high molecular and phenotype heterogeneity. The overall survival rate for gastric cancer is very low because it is always diagnosed in the advanced stages. Therefore, early detection and treatment are of great significance. Currently, biomedical studies have tapped the potential clinical applicability of aptamer-based technology for gastric cancer diagnosis and targeted therapy. Herein, we summarize the enrichment and evolution of relevant aptamers, followed by documentation of the recent developments in aptamer-based techniques for early diagnosis and precision therapy for gastric cancers.

Bright, small sizes and hydro-dispersive NIR persistent luminescence nanoparticles modified with Si and amino groups for enhanced bioimaging.

Near-infrared (NIR) persistent luminescence nanoparticles (PLNPs) with high brightness, small sizes, good hydro-dispersivity, and intrinsic surface-functional groups are desirable in biological applications. In this work, Cr3+-doped zinc gallogermanates Zn1+xGa2-2xGexO4:Cr (ZGGC) PLNPs were hydrothermally synthesized via 3-aminopropyltriethoxysilane (APTES) as an additive, or APTES and cetyltrimethylammonium bromide (CTAB) as two co-additives. Addition of APTES not only dramatically enhances the 696 nm NIR luminescence intensity, but also obviously decreases the particle size and introduces amino groups. In particular, thex= 0.1 series ZGGC (ZGGC0.1) with the addition of n moles equivalent APTES (ZGGC0.1-nA) had smaller particle sizes than thex= 0.2 counterpart (ZGGC0.2-nA). The NIR afterglow intensities increased with the APTES introduction. The ZGGC0.2-2.5A sample (also named as ZGGC, Si, -NH2) exhibited maximum luminescence intensities both in solid and aqueous states. With APTES, Si atom is doped and -NH2groups are modified, the trap depth and density become larger, and the afterglow intensities and decay time are significantly enhanced. More notably, co-addition of CTAB (ZGGC0.2-2.5A-C) (also named as ZGGC, Si, -NH2') further enhances hydro-dispersivity and luminescence intensity, decreases particle sizes, and results in more prominent amino groups. The trap density is drastically higher than that without CTAB (i.e. ZGGC0.2-2.5A). Change of Cr3+microenvironment in the crystal and more defects introduction contribute to the enhanced brightness. As expected, the ZGGC,Si,-NH2' PLNPs possess excellent biocompatibility, deep tissue penetration and distinguished bioimaging properties, and rechargeability with orange LED light. The ZGGC,Si,-NH2' PLNPs should provide to be an excellent nanomaterial for various functionalization and bioimaging applications.

Thermosensitive Biomimetic Hybrid Membrane Camouflaged Hollow Gold Nanoparticles for NIR-Responsive Mild-Hyperthermia Chemo-/Photothermal Combined Tumor Therapy.

As an appealing biomimetic strategy for various medical applications, cell membrane coating lacks sensitive on-demand breaking capability. Herein, we incorporated thermosensitive lipid (TSL) membrane into red blood cell (RBC) and MCF-7 cancer cell (MC) hybrid membrane ([RBC-MC]M) vesicles. The [RBC-MC-TSL]M was coated onto doxorubicin (Dox)-loaded hollow gold nanoparticles to enhance chemo-/photothermal combined tumor therapy at a mild hyperthermia temperature (≤49 °C). Double-layer coating with TSL and [RBC-MC-TSL]M as the inner and outer layer, respectively, presented better antileakage and higher NIR-responsivity than single-layer coating. The Dox release ratio upon NIR laser irradiation (≤49 °C) was 74.6%, much higher than that (33.5%) without NIR laser. The nanodrug can be efficiently and specifically taken up by MCF-7 cells. In addition, the nanodrug exhibited excellent tumor-targeting property, with 4.08- and 1.12-times Dox accumulation in MCF-7 tumors compared to free Dox and [RBC-MC]M-coated counterpart, respectively. Most importantly, TSL incorporation significantly enhanced NIR-responsive antitumor efficiency, with tumor growth inhibition ratio increased from 35.1% to 48.6% after a single dose administration. Besides, the nanodrug exhibited very good biocompatibility. Camouflaging nanoparticles with the thermosensitive biomimetic hybrid membrane provides a painless and promisingly clinical-applicable approach for effective chemo-/photothermal combined mild-hyperthermia tumor therapy.

A DNAzyme-catalyzed label-free aptasensor based on multifunctional dendrimer-like DNA assembly for sensitive detection of carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is an important malign tumor marker. In this study, a simple, label-free and antibody-free aptasensor was fabricated based on a multifunctional dendrimer-like DNA nanoassembly. The DNA nanoassembly was embedded with multiple G-quadruplex DNAzyme motifs and a hanging CEA aptamer motif. It was prepared from short DNA sequences by autonomous-assembly. The aptasensor was prepared simply by self-assembly of a capture DNA (cpDNA) on a gold electrode, followed by hybridization with a CEA aptamer (AptGAC-P). CEA as a model target was detected through competitive binding of CEA with AptGAC-P, exposing cpDNA to bind with the DNA nanoassembly. The detection process only contains 2 incubation steps. The high load of G-quadruplex DNAzyme motifs and their catalytic activity resulted in an amplified and label-free differential pulse voltammetry (DPV) electrochemical signal. The peak current correlated linearly with the CEA concentration, with a linear range of 2-45 ng mL-1, and an LOD value of 0.24 ng mL-1. The aptasensor showed high specificity and reproducibility, and retained 96.5% of detection signal intensities after 31 days of storage. The recovery rates for spiked CEA in human serum were within 100 ± 5%, and the coincidence rates for clinical human serum samples with ELISA kits were 80.7-111%. Conceivably, possessing simplicity, sensitivity, reproducibility, storage stability, and accuracy, the aptasensor should be a very prominent and applicable tool for clinical CEA detection and cancer diagnosis, and is promisingly applicable as a platform for detecting other targets of interests.

Red fluorescent nanoprobe based on Ag@Au nanoparticles and graphene quantum dots for H2O2 determination and living cell imaging.

A sensitive and turn-on fluorescence nanoprobe based on core-shell Ag@Au nanoparticles (Ag@AuNPs) as a fluorescence receptor and red emissive graphene quantum dots (GQDs) as a donor was fabricated. They were conjugated together through π-π stacking between the GQDs and single-strand DNA modified at the Ag@AuNPs surface. The absorption spectrum of the receptor significantly overlapped with the donor emission spectrum, leading to a strong Förster resonance energy transfer (FRET) and thus a dramatic quenching. The sensing mechanism relies on fluorescence recovery following DNA cleavage by •OH produced from Fenton-like reaction between the peroxidase-like Ag nanocore and H2O2. The red emissive feature (Ex/Em, 520 nm/560 nm) provides low background in physiological samples. The •OH production, great spectrum overlapping, and red emission together contributes to good sensitivity and living cell imaging capability. The fluorescence assay (intensity at 560 nm) achieves a low detection limit of 0.49 µM H2O2 and a wide linear range from 5 to 200 µM, superior to most of the reported fluorescent probes. The RSD value for 100 µM H2O2 was 1.4%. The nanoprobe exhibits excellent anti-interferences and shows low cytotoxicity. The recovery of 100 µM standard H2O2 in a cancer cell lysate was 85.8%. Most satisfactorily, it can realize monitoring and imaging H2O2 in living cells. This study not only presents a sensitive H2O2 probe but also provides a platform for detecting other types of reactive oxygen species.

An enzyme-free electrochemical sandwich DNA assay based on the use of hybridization chain reaction and gold nanoparticles: application to the determination of the DNA of Helicobacter pylori.

An ultrasensitive enzyme-free electrochemical sandwich DNA biosensor is described for the detection of ssDNA oligonucleotides. A DNA sequence derived from the genom of Helicobacter pylori was selected as a model target DNA. The DNA assay was realized through catching target DNA on capture DNA immobilized gold electrode; then labeling the target DNA with reporter DNA (rpDNA) and initiator DNA (iDNA) co-modified gold nanoparticles (AuNPs). The high density of iDNAs serves as one of the amplification strategies. The iDNA triggers hybridization chain reaction (HCR) between two hairpins. This leads to the formation of a long dsDNA concatamer strand and represents one amplification strategy. The electrochemical probe [Ru(NH3)5L]2+, where L stands for 3-(2-phenanthren-9-ylvinyl)pyridine, intercalated into dsDNA chain. Multiple probe molecules intercalate into one dsDNA chain, serving as one amplification strategy. The electrode was subjected to differential pulse voltammetry for signal acquisition, and the oxidation peak current at -0.28 V was recorded. On each AuNP, 240 iDNA and 25 rpDNA molecules were immobilized. Successful execution of HCR at the DNA-modified AuNPs was confirmed by gel electrophoresis and hydrodynamic diameter measurements. Introduction of HCR significantly enhances the DNA detection signal intensity. The assay has two linear ranges of different slopes, one from 0.01 fM to 0.5 fM; and one from 1 fM to 100 fM. The detection limit is as low as 0.68 aM. Single mismatch DNA can be differentiated from the fully complementary DNA. Conceivably, this highly sensitive and selective assay provides a general method for detection of various kinds of DNA. Graphical abstractSchematic representation of the detection and the amplification principles of the electrochemical sandwich DNA assay. Purple curl: Captured DNA; Green curl: Reporter DNA; Orange curl: HCR initiator DNA; Yellow solid-circle: Gold nanoparticle; H1 and H2: Two hairpin DNA; [Ru(NH3)5L]2+: Signal probe.

An acetylcholinesterase biosensor based on doping Au nanorod@SiO2 nanoparticles into TiO2-chitosan hydrogel for detection of organophosphate pesticides.

A stable and sensitive electrochemical acetylcholinesterase (AChE) biosensor for detection of organophosphorus pesticides (OPs) was developed by doping Au nanorods (AuNRs)@mesoporous SiO2 (MS) core-shell nanoparticles into CS/TiO2-CS (CS denotes for chitosan) immobilization matrix. AuNRs@MS core-shell nanoparticles were synthesized and characterized. The doping and the biosensor fabrication process were probed and confirmed by scanning electron microscopy and electrochemistry techniques. The doping conditions were optimized. The matrix both before and after AChE immobilization had a mesoporous nanostructure. The nanoparticles dispersed homogeneously within the matrix. The doping significantly enhanced the electro-conductivity of the TiO2-CS hydrogel, and dramatically improved the bioelectrocatalytic activity and OPs detection sensitivity of the AChE immobilized matrix. The detection linear ranges for both dichlovos (DDVP) and fenthion were from 0.018 µM (4.0 ppb) to 13.6 µM, and the limit of detection (LOD) was 5.3 nM (1.2 ppb) and 1.3 nM (0.36 ppb), respectively. The biosensor exhibited high reproducibility and accuracy in detecting OPs spiked vegetable juice samples. In addition, it exhibited very high detection stability and storage stability. The developed AChE biosensor was provided to be a promisingly applicable tool for OPs detection with high reliability, simplicity, and rapidness.

In silico post-SELEX screening and experimental characterizations for acquisition of high affinity DNA aptamers against carcinoembryonic antigen.

DNA aptamers against carcinoembryonic antigen (CEA) have been identified through the systematic evolution of ligands by exponential enrichment (SELEX) technique, but their affinity needs to be improved. In this study, an in silico approach was firstly used to screen the mutation sequences of a reported DNA aptamer (the parent aptamer, denoted as P) against CEA. The affinities of several high-score DNA mutants were determined by the biolayer interferometry technique. Finally, the newly obtained aptamers were verified in an aptasensor application. For the in silico approach, Mfold and RNA Composer were combined to generate the 3D RNA structures of the DNA mutants. The RNA structures were then modified to 3D DNA structures with the Write program. The docking model and binding ability of the 3D DNA structures with CEA were simulated and predicted with the ZDOCK program. Two mutation sequences (P-ATG and GAC-P) exhibited significantly higher ZDOCK scores than P. The dissociation constant of P-ATG and GAC-P to CEA was determined to be 4.62 and 3.93 nM respectively, obviously superior to that of P (6.95 nM). The detection limit of the P-ATG and GAC-P based aptasensors was 1.5 and 1.2 ng mL-1, respectively, markedly better than that based on P (3.4 ng mL-1). The consistency between the in silico and the experimental results indicates that the developed in silico post-SELEX screening approach is feasible for improving DNA aptamers. The P-ATG and GAC-P aptamers found in this study could be used for future CEA aptasensor design and fabrication, promisingly applicable for highly sensitive CEA detection and early cancer diagnosis.

Detection of Helicobacter pylori in dental plaque using a DNA biosensor for noninvasive diagnosis.

Noninvasive diagnosis of Helicobacter pylori (H. pylori) infection is very attractive. This study investigated the single strand DNA (ssDNA) acquisition method from H. pylori in dental plaque, and the integration of our previously developed 43-mer H. pylori DNA biosensor with the obtained target ssDNA (tDNA). Dental plaque samples were collected from 34 patients/volunteers, whose gastric H. pylori infection statuses were tested with the 13C urea breath test (UBT). The samples were treated with colony polymerase chain reaction (PCR) to obtain double strand DNA (dsDNA) of 104 basepairs (bp) long. A blocker ssDNA was designed and used in thermal treatment of the dsDNA to release the 104-mer tDNA, which contains the 43-mer DNA sequence in the middle. PCR primers were designed, and the tDNA releasing and detection conditions with the biosensor were optimized. The limit of detection with the biosensor was 12 fM dsDNA. The dental plaque detection results correlated quite well with the UBT results, with a sensitivity of 100%, and specificity of 97%. These results indicate that the residence of H. pylori in dental plaque is highly associated with gastric H. pylori infection, and detection of dental plaque samples with our DNA biosensor is promisingly applicable in noninvasive diagnosis of H. pylori infection.

Identification of protein tyrosine phosphatase SHP-2 as a new target of perfluoroalkyl acids in HepG2 cells.

Perfluoroalkyl acids (PFAAs) are widespread environmental contaminants which have been detected in humans and linked to adverse health effects. Previous toxicological studies mostly focused on nuclear receptor-mediated pathways and did not support the observed toxic effects. In this study, we aimed to investigate the molecular mechanisms of PFAA toxicities by identifying their biological targets in cells. Using a novel electrochemical biosensor, 16 PFAAs were evaluated for inhibition of protein tyrosine phosphatase SHP-2 activity. Their potency increased with PFAA chain length, with perfluorooctadecanoic acid (PFODA) showing the strongest inhibition. Three selected PFAAs, 25 µM perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid, and PFODA, also inhibited SHP-2 activity in HepG2 cells and increased paxillin phosphorylation level. PFOA was detected in the immunoprecipitated SHP-2 from the cells exposed to 250 µM PFOA, providing unequivocal evidence for the direct binding of PFOA with SHP-2 in the cell. Molecular docking rationalized the formation of PFAA/SHP-2 complex and chain length-dependent inhibition potency. Our results have established SHP-2 as a new cellular target of PFAAs.

In vivo immunotoxicity of perfluorooctane sulfonate in BALB/c mice: Identification of T-cell receptor and calcium-mediated signaling pathway disruption through gene expression profiling of the spleen.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that is used worldwide and is continuously being detected in biota and the environment, thus presenting potential threats to the ecosystem and human health. Although PFOS is highly immunotoxic, its underlying molecular mechanisms remain largely unknown. The present study examined PFOS-induced immunotoxicity in the mouse spleen and explored its underlying mechanisms by gene expression profiling. Oral exposure of male BALB/c mice for three weeks followed by one-week recovery showed that a 10 mg/kg/day PFOS exposure damaged the splenic architecture, inhibited T-cell proliferation in response to mitogen, and increased the percentages of T helper (CD3(+)CD4(+)) and cytotoxic T (CD3(+)CD8(+)) cells, despite the decrease in the absolute number of these cells. A delayed type of PFOS immunotoxicity was observed, which mainly occurred during the recovery period. Global gene expression profiling of mouse spleens and QRT-PCR analyses suggest that PFOS inhibited the expression of genes involved in cell cycle regulation and NRF2-mediated oxidative stress response, and upregulated those in TCR signaling, calcium signaling, and p38/MAPK signaling pathways. Western blot analysis confirmed that the expressions of CAMK4, THEMIS, and CD3G, which were involved in the upregulated pathways, were induced upon PFOS exposure. Acute PFOS exposure modulated calcium homoeostasis in splenocytes. These results indicate that PFOS exposure can activate TCR signaling and calcium ion influx, which provides a clue for the potential mechanism of PFOS immunotoxicity. The altered signaling pathways by PFOS treatment as revealed in the present study might facilitate in better understanding PFOS immunotoxicity and explain the association between immune disease and PFOS exposure.

In vitro immune toxicity of polybrominated diphenyl ethers on murine peritoneal macrophages: apoptosis and immune cell dysfunction.

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and are often detected in the environment, wildlife, and humans, presenting potential threats to ecosystem and human health. PBDEs can cause neurotoxicity, hepatotoxicity, and endocrine disruption. However, data on PBDE immunotoxicity are limited, and the toxicity mechanisms remain largely unknown. Both immune cell death and dysfunction can modulate the responses of the immune system. This study examined the toxic effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and decabromodiphenyl ether (BDE-209) on the immune system by using peritoneal macrophages as the model. The macrophages were exposed to PBDEs, and cell death was determined through flow cytometry and immunochemical blot. The results showed that after 24h of exposure, BDE-47 (>5 µM) and BDE-209 (>20 µM) induced cell apoptosis, increased intracellular reactive oxygen species (ROS) formation and depleted glutathione. BDE-47 was more potent than BDE-209; the cytotoxic concentrations for BDE-47 and BDE-209 were determined to be 5 µM and 20 µM, respectively, during 24h of exposure. However, pretreatment with n-acetyl-l-cysteine (ROS scavenger) partially reversed the cytotoxic effects. Further gene expression analyses on Caspase-3,-8,-9, TNFR1, and Bax revealed that both intrinsic and extrinsic apoptotic pathways were activated. More importantly, non-cytotoxic concentrations BDE-47 (<2 µM) and BDE-209 (<10 µM) could impair macrophage accessory cell function in a concentration-dependent manner, but no effects were observed on phagocytic responses. These revealed effects of PBDEs on macrophages may shed light on the toxicity mechanisms of PBDEs and suggest the necessity of evaluating cellular functionality during the risk assessment of PBDE immunotoxicity.

Structure-activity relations in binding of perfluoroalkyl compounds to human thyroid hormone T3 receptor.

Perfluoroalkyl compounds (PFCs) have been shown to disrupt thyroid functions through thyroid hormone receptor (TR)-mediated pathways, but direct binding of PFCs with TR has not been demonstrated. We investigated the binding interactions of 16 structurally diverse PFCs with human TR, their activities on TR in cells, and the activity of perfluorooctane sulfonate (PFOS) in vivo. In fluorescence competitive binding assays, most of the 16 PFCs were found to bind to TR with relative binding potency in the range of 0.0003-0.05 compared with triiodothyronine (T3). A structure-binding relationship for PFCs was observed, where fluorinated alkyl chain length longer than ten, and an acid end group were optimal for TR binding. In thyroid hormone (TH)-responsive cell proliferation assays, PFOS, perfluorohexadecanoic acid, and perfluorooctadecanoic acid exhibited agonistic activity by promoting cell growth. Furthermore, similar to T3, PFOS exposure promoted expression of three TH upregulated genes and inhibited three TH downregulated genes in amphibians. Molecular docking analysis revealed that most of the tested PFCs efficiently fit into the T3-binding pocket in TR and formed a hydrogen bond with arginine 228 in a manner similar to T3. The combined in vitro, in vivo, and computational data strongly suggest that some PFCs disrupt the normal activity of TR pathways by directly binding to TR.

Single-walled carbon nanotubes and graphene oxides induce autophagosome accumulation and lysosome impairment in primarily cultured murine peritoneal macrophages.

The wide application of carbon nanomaterials in various fields urges in-depth understanding of the toxic effects and underlying mechanisms of these materials on biological systems. Cell autophagy was recently recognized as an important lysosome-based pathway of cell death, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles, but the underlying mechanisms are still uncertain due to the fact that autophagosome accumulation can result from autophagy induction and/or autophagy blockade. In this study, we first evaluated the toxicity of acid-functionalized single-walled carbon nanotubes and graphene oxides, and found that both carbon nanomaterials induced adverse effects in murine peritoneal macrophages, and GOs were more potent than AF-SWCNTs. Both carbon nanomaterials induced autophagosome accumulation and the conversion of LC3-I to LC3-II. However, degradation of the autophagic substrate p62 protein was also inhibited by both nanomaterials. Further analyses on lysosomes revealed that both carbon nanomaterials accumulated in macrophage lysosomes, leading to lysosome membrane destabilization, which indicates reduced autophagic degradation. The effects of AF-SWCNTs and GOs on cell autophagy revealed by this study may shed light on the potential toxic mechanism and suggest caution on their utilization.