Engineering Phage Nanocarriers Integrated with Bio-Intelligent Plasmids for Personalized and Tunable Enzyme Delivery to Enhance Chemodynamic Therapy.

Customizable and number-tunable enzyme delivery nanocarriers will be useful in tumor therapy. Herein, a phage vehicle, T4-Lox-DNA-Fe (TLDF), which adeptly modulates enzyme numbers using phage display technology to remodel the tumor microenvironment (TME) is presented. Regarding the demand for lactic acid in tumors, each phage is engineered to display 720 lactate oxidase (Lox), contributing to the depletion of lactic acid to restructure the tumor's energy metabolism. The phage vehicle incorporated dextran iron (Fe) with Fenton reaction capabilities. H2O2 is generated through the Lox catalytic reaction, amplifying the H2O2 supply for dextran iron-based chemodynamic therapy (CDT). Drawing inspiration from the erythropoietin (EPO) biosynthetic process, an EPO enhancer is constructed to impart the EPO-Keap1 plasmid (DNA) with tumor hypoxia-activated functionality, disrupting the redox homeostasis of the TME. Lox consumes local oxygen, and positive feedback between the Lox and the plasmid promotes the expression of kelch ECH Associated Protein 1 (Keap1). Consequently, the downregulation of the antioxidant transcription factor Nrf2, in synergy with CDT, amplifies the oxidative killing effect, leading to tumor suppression of up to 78%. This study seamlessly integrates adaptable T4 phage vehicles with bio-intelligent plasmids, presenting a promising approach for tumor therapy.

Role of targeting ferroptosis as a component of combination therapy in combating drug resistance in colorectal cancer.

Colorectal cancer (CRC) is a form of cancer that is often resistant to chemotherapy, targeted therapy, radiotherapy, and immunotherapy due to its genomic instability and inflammatory tumor microenvironment. Ferroptosis, a type of non-apoptotic cell death, is characterized by the accumulation of iron and the oxidation of lipids. Studies have revealed that the levels of reactive oxygen species and glutathione in CRC cells are significantly lower than those in healthy colon cells. Erastin has emerged as a promising candidate for CRC treatment by diminishing stemness and chemoresistance. Moreover, the gut, responsible for regulating iron absorption and release, could influence CRC susceptibility through iron metabolism modulation. Investigation into ferroptosis offers new insights into CRC pathogenesis and clinical management, potentially revolutionizing treatment approaches for therapy-resistant cancers.

[Advances in Research on the Mechanism of Association Between Periodontitis and Diabetes Mellitus].

Periodontitis and diabetes mellitus are both chronic diseases with a rather high prevalence and they are closely associated with each other. On one hand, diabetes mellitus poses as a risk factor for periodontitis. On the other hand, periodontitis has a negative impact on glucose control in diabetic patients. The two-way relationship has aroused a lot of research interest in recent years. Herein, approaching the issue by looking at the effect of periodontitis on diabetes, we summarized the mechanism of the traditional periodontal pocket-blood circulation pathway and reviewed the role of the oral-gut axis in the mechanism, which has been proposed in recent years. In addition, regarding the impact of diabetes on periodontitis, we summarized new findings concerning changes in oral microbiota, abnormal levels of cytokines and adipokines, oxidative stress, unbalanced osteogenic and osteoclastic activities, and the accumulation of advanced glycation end-products. We hope this paper will be helpful for further studies on the mechanism of association between periodontitis and diabetes.

Multifunctional nanocarrier with self-catalytic production of nitric oxide for photothermal and gas-combined therapy of tumor.

Single treatment often faces the problem that it cannot completely eradicate tumor and inhibit the tumor metastasis. In order to overcome this shortcoming, multi-modal tumor treatment has attracted widespread attention. In the present article, based on ascorbyl palmitate (PA) and l-arginine (l-Arg), a multifunctional nanocarrier is designed for synergetic treatment of tumor with photothermal and nitric oxide (NO) gas therapy. Firstly, PA and l-Arg were self-assembled to form novel functional micelles, PL, with high biosafety using electrostatic interaction and hydrogen bonding. The functional micelles could self-catalyze to produce NO at the tumor site. Then, Ag2S quantum dots having fluorescence imaging and photothermal properties were encapsulated to obtain the nanocarrier, A@PL. The results show that A@PL had a hydrated size of around 78 nm and presented good stability within 30 d. Moreover, in vitro studies indicate that it was efficient with regards to NO self-generating capacity, whereas the photothermal conversion efficiency was as high as 34% under near-infrared light irradiation. The cytotoxicity results show that, when the concentration of A@PL was as high as 2 mM, the survival rate of 3 T3 cells was still 78.23%, proving that the probe has good safety characteristics. Fluorescence imaging results show that its maximum enrichment can be achieved at the tumor site after tail vein injection for 3 h, and out of the body after 24 h, indicating good internal circulation. The in vivo studies show that the rate of inhibition of tumor using the nanocarrier was as high as 98%, and almost overcame the problem of tumor recurrence caused by single treatment, thus presenting a significant tumor treatment effect. This new multifunctional nanocarrier with self-catalytic production of NO provides a new idea for the efficient treatment of tumors.

Co-delivery of paclitaxel and doxorubicin using polypeptide-engineered nanogels for combination therapy of tumor.

Loading of chemotherapeutic agents into nanoparticles has been demonstrated to be an effective strategy for cancer therapy. However, simultaneous delivery of different functional drugs to tumor sites for chemotherapy still remains challenging. In this study, nanogels formed by an engineered coiled-coil polypeptide PC10A were designed and prepared as a carrier for co-delivery of paclitaxel (PTX) and doxorubicin (DOX) through ultrasonic treatment and electrostatic adsorption. The drug loading content and encapsulation efficiency of PTX and DOX in the PC10A/PTX/DOX nanogels were 5.98 wt%, 70 wt%, and 8.55 wt%, 83 wt%, respectively. Because the polypeptide PC10A was non-toxic and biodegradable, the PC10A/PTX/DOX nanogels exhibited good biocompatibility. Thein vitroandin vivoantitumor experiments showed that the PC10A/PTX/DOX nanogels possessed obviously synergistic therapy effect of tumors and lower side effects compared with free PTX/DOX. Therefore, the PC10A/PTX/DOX nanogels are promising to provide a new strategy for combination therapy of different functional drugs.

A self-assembled nanoplatform based on Ag2S quantum dots and tellurium nanorods for combined chemo-photothermal therapy guided by H2O2-activated near-infrared-II fluorescence imaging.

A nanoplatform based on Ag2S quantum dots (QDs) and tellurium nanorods (TeNRs) was developed for combined chemo-photothermal therapy guided by H2O2-activated near-infrared (NIR)-II fluorescence imaging. Polypeptide PC10AGRD-modified TeNRs and Ag2S QDs were co-encapsulated in 4T1 cell membrane to prepare a nanoplatform (CCM@AT). Ag2S QDs and TeNRs in the CCM@AT were used as a fluorescence probe and photosensitizer, and a chemotherapeutic prodrug and quenching agent to quench the fluorescence of Ag2S QDs, respectively. After the CCM@AT was specifically targeted to the tumor site, the TeNRs were dissolved by the high concentration of H2O2 at the tumor site to light up the fluorescence of Ag2S QDs for NIR-II fluorescence imaging. In addition, the generated toxic TeO66- molecules decreased ATP production by selective cancer chemotherapy, which is beneficial for photothermal therapy. The elevated temperature due to photothermal therapy in turn promoted the chemical reaction in chemotherapy. In vitro and in vivo toxicity results showed that the CCM@AT possesses high biocompatibility. Compared to single photothermal therapy and chemotherapy, the synergistic chemo-photothermal therapy can effectively suppress the growth of 4T1 tumor. This all-in-one nanoplatform provides a boulevard for the combination therapy of tumors guided by NIR-II fluorescence imaging. STATEMENT OF SIGNIFICANCE: NIR-II fluorescence imaging shows the characteristics of low tissue absorption, reflection, and scattering, which can greatly reduce the influence of autofluorescence in vivo. However, the non-negligible effect of autofluorescence is still observed in fluorescence imaging in vivo. Therefore, there is an urgent need to develop a strategy of controlled release of fluorescence for accurate imaging and tumor therapy. Here, Ag2S quantum dots (QDs) with NIR-II fluorescence emission and good photothermal conversion efficiency are used as a fluorescence probe and photosensitizer, and tellurium nanorods (TeNRs) are used as a chemotherapeutic prodrug and quenching agent to quench the fluorescence of Ag2S QDs. This multiple nanoplatform provides an inspiration for the combination therapy of tumor guided by NIR-II fluorescence imaging.

Tumor Microenvironment-Activated Theranostics Nanozymes for Fluorescence Imaging and Enhanced Chemo-Chemodynamic Therapy of Tumors.

Chemodynamic therapy (CDT) is widely explored for tumor-specific therapy by converting endogenous H2O2 to lethal ·OH to destroy cancer cells. However, ·OH scavenging by glutathione (GSH) and insufficient intratumoral H2O2 levels seriously hinder the application of CDT. Herein, we reported the fabrication of copper ion-doped ZIF-8 loaded with gold nanozymes and doxorubicin hydrochloride (DOX) for the chemotherapy and CDT synergistic treatment of tumors with the assistance of tumor microenvironment (TME)-activated fluorescence imaging. The Cu2+-doped ZIF-8 shell was gradually degraded to release DOX and gold nanoclusters responding to the acidic TME. The fluorescence signal of the tumor region was acquired after the quenched fluorescence of the gold nanoclusters by Cu2+ and DOX by aggregation-induced quenching was turned on because of the interaction of GSH with Cu2+ and the release of free DOX. The Cu2+ ions could deplete the GSH via redox reactions and the generated Cu+ could convert internal H2O2 to ·OH for tumor CDT. The chemotherapeutic effect of DOX was strengthened through drug efflux inhibition and drug sensitivity increase due to the consumption of GSH and ·OH burst. Moreover, DOX could raise the level of H2O2 and augment the effect of CDT. In addition, the fluorescent gold nanoclusters not only served as a peroxidase to convert H2O2 to ·OH but also employed as an oxidase to consume GSH, resulting in the amplification of chemotherapy and CDT. This work presents an approach to construct tumor microenvironment-activated theranostic probes without external stimuli and to achieve the tumor elimination through cascade reactions and synergistic treatment.

Within-host model of respiratory virus shedding and antibody response to H9N2 avian influenza virus vaccination and infection in chickens.

Avian influenza virus (AIV) H9N2 subtype is an infectious pathogen that can affect both the respiratory and gastrointestinal systems in chickens and continues to have an important economic impact on the poultry industry. While the host innate immune response provides control of virus replication in early infection, the adaptive immune response aids to clear infections and prevent future invasion. Modelling virus-innate immune response pathways can improve our understanding of early infection dynamics and help to guide our understanding of virus shedding dynamics that could lead to reduced transmission between hosts. While some countries use vaccines for the prevention of H9N2 AIV in poultry, the virus continues to be endemic in regions of Eurasia and Africa, indicating a need for improved vaccine efficacy or vaccination strategies. Here we explored how three type-I interferon (IFN) pathways affect respiratory virus shedding patterns in infected chickens using a within-host model. Additionally, prime and boost vaccination strategies for a candidate H9N2 AIV vaccine are assessed for the ability to elicit seroprotective antibody titres. The model demonstrates that inclusion of virus sensitivity to intracellular type-I IFN pathways results in a shedding pattern most consistent with virus titres observed in infected chickens, and the inclusion of a cellular latent period does not improve model fit. Furthermore, early administration of a booster dose two weeks after the initial vaccine is administered results in seroprotective titres for the greatest length of time for both broilers and layers. These results demonstrate that type-I IFN intracellular mechanisms are required in a model of respiratory virus shedding in H9N2 AIV infected chickens, and also highlights the need for improved vaccination strategies for laying hens.

A within-host mathematical model of H9N2 avian influenza infection and type-I interferon response pathways in chickens.

Chickens infected with avian influenza virus (AIV) transmit the virus via respiratory and cloacal shedding. While previous mathematical models have shown that the innate immune response is necessary for the early suppression of virus production in infected respiratory cells, the different pathways by which the innate immune response can affect cloacal viral shedding have not been studied in chickens. The present study aims to evaluate the sensitivity of H9N2 low pathogenic AIV shedding in chicken gastrointestinal cells to different type-I interferon (IFN) response pathways, and to determine the impact of a cellular eclipse phase (latent period) on the time to peak virus shedding using a mathematical model describing within host viral kinetics. Our model results demonstrate that a mechanistic model that incorporates 1) the intracellular antiviral effects of type-I IFN on virus production, 2) destruction of infected cells by type-I IFN activated Natural Killer cells, and 3) an eclipse phase is most consistent with experimental cloacal virus shedding data. These results provide a potential mechanistic explanation for the delay to peak cloacal virus shedding observed in experimental studies conducted in chickens, as well as an improved understanding of the primary type-I IFN pathways involved in the control of cloacal virus shedding, which may lead to the development of more targeted vaccine candidates.

The effect of forskolin on membrane clock and calcium clock in the hypoxic/reoxygenation of sinoatrial node cells and its mechanism.

BACKGROUND: In this study, we investigated the effect of forskolin (FSK, a selective adenylate cyclase agonist) on the automatic diastolic depolarization of sinus node cells (SNC) with hypoxia/reoxygenation (H/R) injury. METHODS: The SNC of the newborn rat was randomly assigned into the control group, the H/R (H/R injury) group, or the H/R + FSK (H/R injury + FSK treatment) group. Patch-clamp was performed to record the action potential and electrophysiological changes. The cellular distribution of intracellular calcium concentration was analyzed by fluorescence staining. RESULTS: Compared with the control cells, spontaneous pulsation frequency (SPF) and diastolic depolarization rate (DDR) of H/R cells were reduced from 244.3 ± 10.6 times/min and 108.7 ± 7.8 mV/s to 130.5 ± 7.6 times/min and 53.4 ± 6.5 mV/s, respectively. FSK significantly increased SPF and DDR of H/R cells to 208.3 ± 8.3 times/min and 93.2 ± 8.9 mV/s (n = 15, both p < 0.01), respectively. H/R reduced the current densities of If, ICa,T and inward INCX, which were significantly increased by 10 µM FSK treatment (n = 15, p < 0.01). Furthermore, reduced expression of HCN4 and NCX1.1 channel protein were significantly increased by FSK. Inhibitor studies showed that both SQ22536 (a selective adenylate cyclase inhibitor) and H89 (a selective protein kinases A [PKA] inhibitor) blocked the effects of FSK on SPF and DDR. CONCLUSIONS: H/R causes pacemaker dysfunction in newborn rat sinoatrial node cells leading to divergence of the DD and the slow of spontaneous APs, which change can be dramatically reversed by FSK through increasing INCX and If current in H/R injury.

Inhibitory Effects of Cyclopiazonic Acid on the Pacemaker Current in Sinoatrial Nodal Cells.

OBJECTIVE: The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells. METHODS: at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. If and HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis. RESULTS: The diastolic depolarization rate of spontaneous action potentials and the current densities of If were reduced by exposure to 10 µM CPA. The inhibitory effect of CPA was concentration-dependent with an IC50 value of 16.3 µM and a Hill coefficient of 0.98. The effect of CPA on If current was also time-dependent, and the If current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the If current was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA. CONCLUSIONS: These results indicate that the direct inhibition effect of CPA on the If current in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression.

Prevalence of fecal viruses and bacteriophage in Canadian farmed mink (Neovison vison).

Recent viral metagenomic studies have demonstrated the diversity of eukaryotic viruses and bacteriophage shed in the feces of domestic species. Although enteric disease is a major concern in the commercial mink farming industry, few etiologic agents have been well characterized. This study aimed to identify viruses shed in the fecal matter of clinically healthy commercial mink from 40 southern Ontario farms. Viral RNA was extracted from 67 pooled fecal samples (30 adult female mink and 37 kit) and amplified for Illumina sequencing on the NextSeq platform, and the resulting contigs were trimmed and assembled using Trimmomatic 0.36.0 and Spades 3.8.0 in iVirus (CyVerse, AZ, USA) and SeqMan NGen 12 (DNAStar, WI, USA). Identification of assembled sequences >100 bp (Geneious 10.1.3) showed an abundance of bacteriophage sequences, mainly from families Siphoviridae (53%), Podoviridae (22%), Myoviridae (20%), Inoviridae (1%), Leviviridae (0.04%), Tectiviridae (0.01%), and Microviridae (0.01%). A diverse range of vertebrate viruses were detected, of which posavirus 3, mink bocavirus, gyroviruses, and avian-associated viruses were most abundant. Additionally, sequences from nonvertebrate viruses with water and soil-associated amebal and algal hosts were also highly prevalent. The results of this study show that viruses shed in the fecal matter of healthy commercial mink are highly diverse and could be closely associated with diet, and that more research is necessary to determine how the detected viruses may impact mink health.

Calcium-Mediated Oscillation in Membrane Potentials and Atrial-Triggered Activity in Atrial Cells of Casq2R33Q/R33Q Mutation Mice.

Aim: We investigated the underlying mechanisms in atrial fibrillation (AF) associated with R33Q mutation and Ca2+-triggered activity. Methods and Results: We examined AF susceptibility with intraesophageal burst pacing in the sarcoplasmic reticulum (SR) Ca2+ leak model calsequestrin 2 R33Q (Casq2R33Q/R33Q) mice. Atrial trigger appeared in R33Q mice but not WT mice (17.24%, 5/29 vs. 0.00%, 0/32, P < 0.05). AF was induced by 25 Hz pacing in R33Q mice (48.27%, 14/29 vs. 6.25%, 2/32, P < 0.01). The mice were given 1.5 mg/kg isoproterenol (Iso), and the incidences of AF increased (65.51%, 19/29 vs. 9.21%, 3/32, P < 0.01). Electrophysiology experiments and the recording of intracellular Ca2+ indicated significant increases in the Ca2+ sparks (5.24 ± 0.75 100 µM-1.s-1 vs. 0.29 ± 0.04 100 µM-1.s-1, n = 20, P < 0.05), intracellular free Ca2+ (0.238 ± 0.009 µM vs. 0.172 ± 0.006 µM, n = 20, P < 0.05), Ca2+ wave (11.74% vs. 2.24%, n = 20, P < 0.05), transient inward current (ITi) (-0.56 ± 0.02 pA/pF vs. -0.42 ± 0.01 pA/pF, n = 10, P < 0.05), and oscillation in membrane potentials (10.71%, 3/28 vs. 4.16%, 1/24, P < 0.05) in the R33Q group, but there was no significant difference in the L-type calcium current. These effects were enhanced by Iso, and the inhibition of calmodulin-dependent protein kinase II (CaMKII) by 1 µM KN93 reversed the effects of Iso on Ca2+ sparks (5.01 ± 0.66 100 µm-1.s-1 vs. 11.33 ± 1.63 100 µm-1.s-1, P < 0.05), intracellular Ca2+ (0.245 ± 0.005 µM vs. 0.324 ± 0.008 µM, P < 0.05), Ca2+ wave (12.35% vs. 17.83%, P < 0.05), ITi (-0.61 ± 0.02 pA/pF vs. -0.78 ± 0.03 pA/pF, n = 10, P < 0.05), and oscillation in membrane potential (17.85% 5/28 vs. 32.17% 9/28, P < 0.05). The reduction of ryanodine receptor 2 (RyR2) stable subunits (Casq2, triadin, and junctin) rather than RYR2 and the increase in CaMKII, phosphor-CaMKII, phosphor-RyR2 (Ser 2814), SERCA, and NCX1.1 was reflected in the R33Q group. Conclusion: This study demonstrates that the increase in spontaneous calcium elevations corresponding to ITi that may trigger the oscillation in membrane potentials in the R33Q group, thereby increasing the risk of AF. The occurrence of spontaneous calcium elevations in R33Q atrial myocytes is due to the dysfunction of RyR2 stable subunits, CaMKII hyperactivity, and CaMKII-mediated RyR phosphorylation. An effective therapeutic strategy to intervene in Ca2+-induced AF associated with the R33Q mutation may be through CaMKII inhibition.

Detection of Astrovirus, Rotavirus C, and Hepatitis E Viral RNA in Adult and Juvenile Farmed Mink (Neovison vison).

Mink astrovirus (MiAstV) is known to play a major role in mink pre-weaning diarrhea, and rotavirus and hepatitis E virus (HEV) are both considered potentially zoonotic agents. These viruses are not monitored in commercial mink, and the role of these viral infections in mink health is not well understood. This study assessed the prevalence of mink astrovirus, rotavirus C, mink HEV and swine HEV in 527 pooled healthy adult female mink and mink kit fecal samples from 50 Canadian mink farms in two seasons over 4 years. Viral RNA was extracted and amplified in RT-PCR to detect mink astrovirus and HEV RdRp genes, swine HEV ORF2, and rotavirus C VP6 gene. At least 26% of all positive samples for each virus was sequenced for phylogenetic analysis. Fourteen percent of samples were astrovirus positive, while 3 and 9% of samples were rotavirus C and mink HEV positive, respectively. One adult female sample was found to be positive by PCR for swine HEV. A significantly higher number of kit samples were astrovirus- and HEV-positive compared to adult female samples (p = 0.01 and p < 0.0001, respectively). Astrovirus was detected in significantly more summer samples from adult females compared to winter samples from adult females (p = 0.001). The detected sequences were closely related to previously reported MiAstV, swine rotavirus C, and mink and swine HEV strains. Two astrovirus sequences were distantly related to all other detected sequences as well as previously reported MiAstVs. These results demonstrate low to moderate prevalence of the three viruses in feces from clinically healthy Canadian commercial mink, and suggest that further monitoring of these viruses may provide a better understanding of how these potentially zoonotic agents may play a role in mink enteritis and overall productivity.

[The reverse effects of allitridum on sodium current decrease caused by SCN5A-F1473S mutation].

This study was designed to test the allitridum (All) activity in correction of sodium current decrease caused by SCN5A-F1473S mutation in HEK293 cells. The result may provide a theoretical basis for screening of new drugs in the treatment of Brugada syndrome. We transferred SCN5A-F1473S channel plasmids into HEK293 cells in a transient transfection. All was administrated acutely and chronically using extracellular irrigation flow and co-culture model. The concentration of All was 30 µmol·L(-1). We used whole cell patch clamp technique in voltage clamp mode to record current and gating kinetics. In order to explore the rescue function of All on decreased sodium peak current, we used confocal microscopy and Western blot to detect the expression of channel proteins in the cell membrane. We found a significant increase in sodium peak current of the 30 µmol·L(-1) All HEK293 cells (269.8 ± 16.6 pA/pF, P < 0.01), almost closed to the current density of the control group（298.2 ± 17.5 p A/p F, P < 0.01）. All allowed the steady-state inactivation of the channel to move toward a more positive direction (V(1/2, inact) returns to -79.5 ± 2.4 mV, P < 0.01). It also slowed the intermediate state inactivation of the channel (inactivation prolongated to 598.1 ± 22.6 ms, P < 0.01). Meanwhile, All increased distribution and expression of the channel protein in the cell membrane (compared to F1473S, P < 0.01). All caused an increase of current in SCN5A-F1473S mutation cells. We consider that the main mechanism may be related to the reduced channel inactivation by the drug with an improvement of the migration barrier of the mutational channel.