Enhanced In Vitro Antiviral Activity of Ivermectin-Loaded Nanostructured Lipid Carriers against Porcine Epidemic Diarrhea Virus via Improved Intracellular Delivery.

Porcine epidemic diarrhea virus (PEDV) is an acute enteric coronavirus, inducing watery diarrhea and high mortality in piglets, leading to huge economic losses in global pig industry. Ivermectin (IVM), an FDA-approved antiparasitic agent, is characterized by high efficacy and wide applicability. However, the poor bioavailability limits its application. Since the virus is parasitized inside the host cells, increasing the intracellular drug uptake can improve antiviral efficacy. Hence, we aimed to develop nanostructured lipid carriers (NLCs) to enhance the antiviral efficacy of IVM. The findings first revealed the capacity of IVM to inhibit the infectivity of PEDV by reducing viral replication with a certain direct inactivation effect. The as-prepared IVM-NLCs possessed hydrodynamic diameter of 153.5 nm with a zeta potential of -31.5 mV and high encapsulation efficiency (95.72%) and drug loading (11.17%). IVM interacted with lipids and was enveloped in lipid carriers with an amorphous state. Furthermore, its encapsulation in NLCs could enhance drug internalization. Meanwhile, IVM-NLCs inhibited PEDV proliferation by up to three orders of magnitude in terms of viral RNA copies, impeding the accumulation of reactive oxygen species and mitigating the mitochondrial dysfunction caused by PEDV infection. Moreover, IVM-NLCs markedly decreased the apoptosis rate of PEDV-induced Vero cells. Hence, IVM-NLCs showed superior inhibitory effect against PEDV compared to free IVM. Together, these results implied that NLCs is an efficient delivery system for IVM to improve its antiviral efficacy against PEDV via enhanced intracellular uptake.

Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

Canine mammary tumors (CMT) can severely compromise the life quality of the affected dogs through local recurrence, distant metastases and ultimately succumb to death. Recently, more attention has been given to the potential antimetastatic effect of maduramicin (MAD) on breast cancer. However, its poor aqueous solubility and toxicity to normal tissues limit its clinical application. Therefore, to address the drawbacks of MAD and enhance its anticancer and antimetastatic effects, MAD-loaded TPGS polymeric micelles (MAD-TPGS) were prepared by a thin-film hydration technique. The optimized MAD-TPGS exhibited excellent size distribution, stability and improved water solubility. Cellular uptake assays showed that TPGS polymer micelles could enhance drug internalization. Moreover, TPGS synergistically improved the cytotoxicity of MAD by targeting mitochondrial organelles, improving reactive oxygen species levels and reducing the mitochondrial transmembrane potential. More importantly, MAD-TPGS significantly impeded the metastasis of tumor cells. In vivo results further confirmed that, in addition to exhibiting excellent biocompatibility, MAD-TPGS exhibited greater antitumor efficacy than free MAD. Interestingly, MAD-TPGS displayed superior suppression of CMT metastasis via tail vein injection compared to oral administration, indicating its suitability for intravenous delivery. Overall, MAD-TPGS could be applied as a potential antimetastatic cancer agent for CMT.

Methyl halide transferase-based gas reporters for quantification of filamentous bacteria in microdroplet emulsions.

The application of microfluidic techniques in experimental and environmental studies is a rapidly emerging field. Water-in-oil microdroplets can serve readily as controllable micro-vessels for studies that require spatial structure. In many applications, it is useful to monitor cell growth without breaking or disrupting the microdroplets. To this end, optical reporters based on color, fluorescence, or luminescence have been developed. However, optical reporters suffer from limitations when used in microdroplets such as inaccurate readings due to strong background interference or limited sensitivity during early growth stages. In addition, optical detection is typically not amenable to filamentous or biofilm-producing organisms that have significant nonlinear changes in opacity and light scattering during growth. To overcome such limitations, we show that volatile methyl halide gases produced by reporter cells expressing a methyl halide transferase (MHT) can serve as an alternative nonoptical detection approach suitable for microdroplets. In this study, an MHT-labeled Streptomyces venezuelae reporter strain was constructed and characterized. Protocols were established for the encapsulation and incubation of S. venezuelae in microdroplets. We observed the complete life cycle for S. venezuelae including the vegetative expansion of mycelia, mycelial fragmentation, and late-stage sporulation. Methyl bromide (MeBr) production was detected by gas chromatography-mass spectrometry (GC-MS) from S. venezuelae gas reporters incubated in either liquid suspension or microdroplets and used to quantitatively estimate bacterial density. Overall, using MeBr production as a means of quantifying bacterial growth provided a 100- to 1,000-fold increase in sensitivity over optical or fluorescence measurements of a comparable reporter strain expressing fluorescent proteins. IMPORTANCE Quantitative measurement of bacterial growth in microdroplets in situ is desirable but challenging. Current optical reporter systems suffer from limitations when applied to filamentous or biofilm-producing organisms. In this study, we demonstrate that volatile methyl halide gas production can serve as a quantitative nonoptical growth assay for filamentous bacteria encapsulated in microdroplets. We constructed an S. venezuelae gas reporter strain and observed a complete life cycle for encapsulated S. venezuelae in microdroplets, establishing microdroplets as an alternative growth environment for Streptomyces spp. that can provide spatial structure. We detected MeBr production from both liquid suspension and microdroplets with a 100- to 1,000-fold increase in signal-to-noise ratio compared to optical assays. Importantly, we could reliably detect bacteria with densities down to 106 CFU/mL. The combination of quantitative gas reporting and microdroplet systems provides a valuable approach to studying fastidious organisms that require spatial structure such as those found typically in soils.

Impact of Environmental Regulation on Carbon Emissions in Countries along the Belt and Road-An Empirical Study Based on PSTR Model.

Since China has put forward the Belt and Road Initiative in 2013, research on the BRI-related countries along the Belt and Road has sprung up. With the advent of the era of carbon peak and carbon neutralization, environmental regulation, as one of the important methods to control carbon emissions, is becoming increasingly prominent. Research on the impact pathway of environmental regulation of countries along the Belt and Road on carbon emissions has important implications for environmental protection and carbon emission reduction. Based on the panel data of 38 countries along the Belt and Road from 2005 to 2018, this research applied linear Tobit model and nonlinear dynamic panel regression model (PSTR) to evaluate the direct impacts on carbon emissions from environmental regulation of countries along the Belt and Road, and to analyze the different impacts of environmental regulation on carbon emissions in terms of technical innovation, industrial structure, and energy intensity. We found that (1) the direct impact of environmental regulation on carbon emissions in the countries along the Belt and Road is positive, with slight differences between the Silk Road Economic Belt and 21st Century Maritime Silk Road (2) when technical innovation is at a low level, environmental regulation promotes carbon emissions, while at a high level, environmental regulation significantly inhibits carbon emissions. (3) When industrial structure is at both a low and high level, environmental regulation inhibits carbon emissions, with a stronger degree of inhibition at a higher level. (4) When energy intensity is at a low level, environmental regulation promotes carbon emissions, while at a high level, environmental regulation inhibits carbon emissions. Accordingly, we suggest that countries along the Belt and Road follow the road of sustainable and low-carbon development, which should further enhance their focus on environment protection, improve their environmental awareness, and take environmental regulation measures rationally to reduce carbon emissions. Meanwhile, relevant adjustments should be done on technical innovation, industrial structure, and energy intensity to achieve carbon emission reduction.

Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA-MurG interaction.

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

Nano-encapsulation of halofuginone hydrobromide enhances anticoccidial activity against Eimeria tenella in chickens.

Coccidiosis is a worldwide epidemic intestinal disease with high incidence, which causes huge economic losses. Halofuginone hydrobromide (HF) is widely applied as an effective anticoccidial drug in the poultry industry. However, its therapeutic efficacy is severely restrained due to toxic effects, poor aqueous solubility and low permeability. Nanotechnology can improve the biological effect of drugs, and thus, reduce administered doses and toxic effects. The objective of this study was to investigate the therapeutic and preventive potential of novel HF-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles (HTPM) for preventing coccidiosis in chickens. The HTPM were approximately spherical with a hydrodynamic diameter of 12.65 ± 0.089 nm, a zeta potential of 8.03 ± 0.242 mV, a drug loading of 14.04 ± 0.12%, and an encapsulation efficiency of 71.1 ± 4.15%. HF was encapsulated in the polymer micelles through interactions with TPGS, as characterized by X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. Cellular take up assays showed that TPGS polymer micelles could enhance drug internalization to alleviate intestinal apoptosis induced by coccidiosis and promote the necrosis of second-generation merozoites of E. tenella. Notably, clinical trials proved that 1.5 mg L-1 HTPM had a stronger anticoccidial effect on E. tenella than that of 3 mg kg-1 HF premix. Amplicon sequencing identified that HTPM could alleviate coccidiosis by restoring the structure of the gut microbiome. These findings indicated that the anticoccidial efficacy of HF was significantly enhanced after being encapsulated in polymer micelles, and further demonstrated the potential protective application of nano-encapsulating anticoccidial drugs as a promising approach to control coccidiosis in poultry. In summary, HTPM hold huge potential as an effective therapeutic agent for coccidiosis.

Intracellular Experimental Evolution of Francisella tularensis Subsp. holarctica Live Vaccine Strain (LVS) to Antimicrobial Resistance.

In vitro experimental evolution has complemented clinical studies as an excellent tool to identify genetic changes responsible for the de novo evolution of antimicrobial resistance. However, the in vivo context for adaptation contributes to the success of particular evolutionary trajectories, especially in intracellular niches where the adaptive landscape of virulence and resistance are strongly coupled. In this work, we designed an ex vivo evolution approach to identify evolutionary trajectories responsible for antibiotic resistance in the Live Vaccine Strain (LVS) of Francisella tularensis subsp. holarctica while being passaged to increasing ciprofloxacin (CIP) and doxycycline (DOX) concentrations within macrophages. Overall, adaptation within macrophages advanced much slower when compared to previous in vitro evolution studies reflecting a limiting capacity for the expansion of adaptive mutations within the macrophage. Longitudinal genomic analysis identified resistance conferring gyrase mutations outside the Quinolone Resistance Determining Region. Strikingly, FupA/B mutations that are uniquely associated with in vitro CIP resistance in Francisella were not observed ex vivo, reflecting the coupling of intracellular survival and resistance during intracellular adaptation. To our knowledge, this is the first experimental study demonstrating the ability to conduct experimental evolution to antimicrobial resistance within macrophages. The results provide evidence of differences in mutational profiles of populations adapted to the same antibiotic in different environments/cellular compartments and underscore the significance of host mediated stress during resistance evolution.

Analysis of Left Atrial Function after Percutaneous Intramyocardial Septal Radiofrequency Ablation in Hypertrophic Cardiomyopathy.

INTRODUCTION: The main goal of our research was to explore the effect of percutaneous intramyocardial septal radiofrequency ablation (PIMSRA) on left atrial (LA) phasic function in hypertrophic cardiomyopathy (HCM). METHODS: The study included 13 patients who underwent PIMSRA at our hospital. The function of LA including reservoir, conduit, and booster pump was analyzed and compared before and 6 months after PIMSRA in HCM patients. LA reservoir function parameters contain maximal LA volume, minimal LA volume (LAV min), LA ejection fraction (LAEF), LA expansion index (LAEI), and reservoir strain; LA conduit function includes LA volume before atrial systole, LA passive volume, LA passive ejection fraction, and conduit strain; LA booster function involves LA booster volume, LA active ejection fraction, and LA contraction strain. Additionally, 20 healthy controls were selected to compare the LA function of HCM patients. RESULTS: The preoperative LA reservoir and conduit function in HCM patients were significantly impaired compared with the control group, while the change in booster pump function was not obvious. HCM patients at 6 months after PIMSRA had remarkably enhanced reservoir and conduit functions which were manifested by lower LAV min, higher LAEF, LAEI, reservoir, and conduit strain than before the operation, and the differences among these parameters between patients after PIMSRA and the healthy control group were not significant. However, with regard to LA contraction function, there was no significant improvement at 6 months after PIMSRA compared with before operation. CONCLUSION: PIMSRA is effective in the amelioration of LA reservoir and conduit function in patients with HCM but not in a marked improvement of LA contraction function in these individuals in short term.

Repurposing maduramicin as a novel anticancer and anti-metastasis agent for triple-negative breast cancer as enhanced by nanoemulsion.

Triple-negative breast cancer (TNBC) is featured by aggression and metastasis and remains an unmet medical challenge due to high death rate. We aimed to repurpose maduramicin (MAD) as an effective drug against TNBC, and develop a nanoemulsion system to enhance anticancer efficacy of MAD. MDA-MB-231 and 4 T1 cells were used as in vitro model, and cell viability was determined by performing cell counting kit-8 and a colony-formation assay. Furthermore, MAD loaded nanoemulsion (MAD-NEs) was manufactured and characterized by a series of tests. The anticancer and anti-metastasis mechanism of MAD-NEs were assessed by performing cell cycle, apoptosis, wound-healing, transwell assay and Western blotting assays. Herein, MAD was firstly demonstrated to be an effective agent to suppress growth of TNBC cells. Subsequently, the optimized MAD-NEs were shown to have stability and high encapsulation efficiency, and could arrested cells in G0/G1 phase and induced apoptosis in TNBC cells. More importantly, MAD-NEs significantly impeded the metastasis of tumor cells, which was further demonstrated by the significant altered expression of epithelial-mesenchymal transition and extracellular matrix markers in vitro and in vivo. Moreover, compared to MAD, MAD-NEs exhibited higher efficacy in shrinking breast tumor size and repressing liver and lung metastasis in vivo, and showed excellent biocompatibility in tumor-bearing mice. The successfully prepared MAD-NEs are expected to be harnessed to suppress tumor growth, invasion and metastasis in the battle against malignant TNBC.

Optimization of Maduramicin Ammonium-Loaded Nanostructured Lipid Carriers Using Box-Behnken Design for Enhanced Anticoccidial Effect against Eimeria tenella in Broiler Chickens.

Maduramicin ammonium (MAD) is one of the most frequently used anticoccidial agents in broiler chickens. However, the high toxicity and low solubility of MAD limit its clinical application. In this study, MAD-loaded nanostructured lipid carriers (MAD-NLCs) were prepared to overcome the defects of MAD by using highly soluble nanostructured lipid carriers (NLCs). The formulation was optimized via a three-level, three-factor Box-Behnken response surface method. Then, the optimal MAD-NLCs were evaluated according to their hydrodynamic diameter (HD), zeta potential (ZP), crystal structure, encapsulation efficiency (EE), drug loading (DL), in vitro release, and anticoccidial effect. The optimal MAD-NLCs had an HD of 153.6 ± 3.044 nm and a ZP of -41.4 ± 1.10 mV. The X-ray diffraction and Fourier-transform infrared spectroscopy results indicated that the MAD was encapsulated in the NLCs in an amorphous state. The EE and DL were 90.49 ± 1.05% and 2.34 ± 0.04%, respectively, which indicated that the MAD was efficiently encapsulated in the NLCs. In the in vitro study, the MAD-NLCs demonstrated a slow and sustained drug release behavior. Notably, MAD-NLCs had an excellent anticoccidial effect against Eimeria tenella in broiler chickens. In summary, MAD-NLCs have huge potential to form a new preparation administered via drinking water with a powerful anticoccidial effect.

Orally Administered Halofuginone-Loaded TPGS Polymeric Micelles Against Triple-Negative Breast Cancer: Enhanced Absorption and Efficacy with Reduced Toxicity and Metastasis.

Background: Halofuginone (HF)-loaded TPGS polymeric micelles (HTPM) were successfully fabricated using the thin-film hydration technique. HTPM via intravenous injection have been demonstrated to exert an excellent anticancer effect against triple-negative breast cancer (TNBC) cells and subcutaneous xenografts. In the present study, we further explored the potential treatment effect and mechanism of orally administered HTPM alone and in combination with surgical therapy on TNBC in subcutaneous and orthotopic mouse models. Methods: Herein, the stability and in vitro release behavior of HTPM were first evaluated in the simulated gastrointestinal fluids. Caco-2 cell monolayers were then used to investigate the absorption and transport patterns of HF with/without encapsulation in TPGS polymeric micelles. Subsequently, the therapeutic effect of orally administered HTPM was checked on subcutaneous xenografts of TNBC in nude mice. Ultimately, orally administered HTPM, combined with surgical therapy, were utilized to treat orthotopic TNBC in nude mice. Results: Our data confirmed that HTPM exhibited good stability and sustained release in the simulated gastrointestinal fluids. HF was authenticated to be a substrate of P-glycoprotein (P-gp), and its permeability across Caco-2 cell monolayers was markedly enhanced via heightening intracellular absorption and inhibiting P-gp efflux due to encapsulation in TPGS polymeric micelles. Compared with HF alone, HTPM showed stronger tumor-suppressing effects in subcutaneous xenografts of MDA-MB-231 cells when orally administered. Moreover, compared with HTPM or surgical therapy alone, peroral HTPM combined with partial surgical excision synergistically retarded the growth of orthotopic TNBC. Fundamentally, HTPM orally administered at the therapeutic dose did not cause any pathological injury, while HF alone led to weight loss and jejunal bleeding in the investigated mice. Conclusion: Taken together, HTPM could be applied as a potential anticancer agent for TNBC by oral administration.

Microfluidic platform for spatially segregated experimental evolution studies with E. coli.

Microdroplet emulsions allow investigators to build controllable microenvironments for applications in experimental evolution and synthetic ecology. We designed a microfluidic platform that uses highly homogenous microdroplets to enable these experiments. We also present a step-by-step protocol for the rapid production of highly homogeneous microdroplets suitable for experimental evolution. We also describe protocols for the propagation and serial passage of microbial populations across a range of selection schemes and potential spatial structures. For complete details on the use and execution of this protocol, please refer to Seo et al. (2021).

Identification of Evolutionary Trajectories Associated with Antimicrobial Resistance Using Microfluidics.

In vitro experimental evolution of pathogens to antibiotics is commonly used for the identification of clinical biomarkers associated with antibiotic resistance. Microdroplet emulsions allow exquisite control of spatial structure, species complexity, and selection microenvironments for such studies. We investigated the use of monodisperse microdroplets in experimental evolution. Using Escherichia coli adaptation to doxycycline, we examined how changes in environmental conditions such as droplet size, starting lambda value, selection strength, and incubation method affected evolutionary outcomes. We also examined the extent to which emulsions could reveal potentially new evolutionary trajectories and dynamics associated with antimicrobial resistance. Interestingly, we identified both expected and unexpected evolutionary trajectories including large-scale chromosomal rearrangements and amplification that were not observed in suspension culture methods. As microdroplet emulsions are well-suited for automation and provide exceptional control of conditions, they can provide a high-throughput approach for biomarker identification as well as preclinical evaluation of lead compounds.

Maduramicin induces cardiotoxicity via Rac1 signaling-independent methuosis in H9c2 cells.

Maduramicin frequently induces severe cardiotoxicity in target and nontarget animals in clinic. Apoptotic and non-apoptotic cell death mediate its cardiotoxicity; however, the underlying non-apoptotic cell death induced by maduramicin remains unclear. In current study, a recently described non-apoptotic cell death "methuosis" caused by maduramicin was defined in mammalian cells. Rat myocardial cell H9c2 was used as an in vitro model, showing excessively cytoplasmic vacuolization upon maduramicin (0.0625-5 µg/mL) exposure for 24 h. Maduramicin-induced reversible cytoplasmic vacuolization of H9c2 cells in a time- and concentration-dependent manner. The vacuoles induced by maduramicin were phase lucent with single membrane and were not derived from the swelling of organelles such as mitochondria, endoplasmic reticulum, lysosome, and Golgi apparatus. Furthermore, maduramicin-induced cytoplasmic vacuoles are generated from micropinocytosis, which was demonstrated by internalization of extracellular fluid-phase marker Dextran-Alexa Fluor 488 into H9c2 cells. Intriguingly, these cytoplasmic vacuoles acquired some characteristics of late endosomes and lysosomes rather than early endosomes and autophagosomes. Vacuolar H+ -ATPase inhibitor bafilomycin A1 efficiently prevented the generation of cytoplasmic vacuoles and decreased the cytotoxicity of H9c2 cells triggered by maduramicin. Mechanism studying indicated that maduramicin activated H-Ras-Rac1 signaling pathway at both mRNA and protein levels. However, the pharmacological inhibition and siRNA knockdown of Rac1 rescued maduramicin-induced cytotoxicity of H9c2 cells but did not alleviate cytoplasmic vacuolization. Based on these findings, maduramicin induces methuosis in H9c2 cells via Rac-1 signaling-independent seriously cytoplasmic vacuolization.

Encapsulating Halofuginone Hydrobromide in TPGS Polymeric Micelles Enhances Efficacy Against Triple-Negative Breast Cancer Cells.

BACKGROUND: Halofuginone hydrobromide (HF) is a synthetic analogue of the naturally occurring quinazolinone alkaloid febrifugine, which has potential therapeutic effects against breast cancer, however, its poor water solubility greatly limits its pharmaceutical application. D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of vitamin E, which can self-assemble to form polymeric micelles (PMs) for encapsulating insoluble anti-tumor drugs, thereby effectively enhancing their anti-cancer effects. METHODS: HF-loaded TPGS PMs (HTPMs) were manufactured using a thin-film hydration technique, followed by a series of characterizations, including the hydrodynamic diameter (HD), zeta potential (ZP), stability, drug loading (DL), encapsulation efficiency (EE), and in vitro drug release. The anti-cancer effects and potential mechanism of HTPMs were investigated in the breast cell lines MDA-MB-231 and MCF-7, and normal breast epithelial cell line Eph-ev. The breast cancer-bearing BALB/c nude mouse model was successfully established by subcutaneous injection of MDA-MB-231 cells and used to evaluate the in vivo therapeutic effect and safety of the HTPMs. RESULTS: The optimized HTPMs had an HD of 17.8±0.5 nm and ZP of 14.40±0.1 mV. These PMs exhibited DL of 12.94 ± 0.46% and EE of 90.6 ± 0.85%, along with excellent storage stability, dilution tolerance and sustained drug release in pH-dependent manner within 24 h compared to free HF. Additionally, the HTPMs had stronger inhibitory effects than free HF and paclitaxel against MDA-MB-231 triple-negative breast cancer cells, and little toxicity in normal breast epithelial Eph-ev cells. The HTPMs induced cell cycle arrest and apoptosis of MDA-MB-231 by disrupting the mitochondrial membrane potential and enhancing reactive oxygen species formation. Evaluation of in vivo anti-tumor efficacy demonstrated that HTPMs exerted a stronger tumor inhibition rate (68.17%) than free HF, and exhibited excellent biocompatibility. CONCLUSION: The findings from this study indicate that HTPMs holds great clinical potential for treating triple-negative breast cancer.

Effect of maduramicin on crayfish (Procambius clarkii): Hematological parameters, oxidative stress, histopathological changes and stress response.

Maduramicin, an extensively used anticoccidial drug, has been introduced into environment due to poorly absorbed in the intestine of broiler chicken. To understand the potential ecological toxicity of maduramicin on aquatic organisms, acute and subacute toxicity, hemolymph biochemistry, histopathology and the expressions of drug metabolism and stress response genes of crayfish (Procambius clarkii) were investigated in this study. For the first time, the 96 h median lethal concentration (LC50) of maduramicin on crayfish was 67.03 mgL-1 with a 95% confidence interval (54.06-81.32 mgL-1). Then, the crayfish were exposed to 0.7 mgL-1 (1/100 LC50), 3.5 mgL-1 (1/20 LC50) and 7.0 mgL-1 (1/10 LC50) maduramicin for 28 days. Maduramicin significantly altered biochemical parameters including AST, ALT, CK, LDH and ALP of hemolymph in crayfish at several time points. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) of crayfish gills, hepatopancreas and abdominal muscle were significantly decreased or elevated by different concentrations of maduramicin treatment at varying time points. Furthermore, histopathological damage of crayfish gills, hepatopancreas and abdominal muscle were observed in a concentration-dependent manner. The expressions of metabolic and stress response genes (CYP450, GST, COX1, COX2, HSP70 and MT) in hepatopancreas of crayfish were significantly up-regulated by maduramicin (7.0 mgL-1) treatment for 8 h to 7 d, and returned to normal levels after the removal of maduramicin for 3-7 days. In conclusion, our findings demonstrated that environmental exposure of maduramicin threaten to the health of crayfish living in the areas nearby livestock farms or pharmaceutical factory. Crayfish exhibited resistance to the stress of maduramicin via activating drug metabolite and detoxification pathways.

Ionophore Toxin Maduramicin Produces Haff Disease-Like Rhabdomyolysis in a Mouse Model.

Maduramicin is a toxic ionophore antibiotic that is isolated from Streptomyces, frequently occurring in an aquatic environment. To understand the potential role of maduramicin in crayfish consumption related Haff disease, a mouse model was established in this study. Two exposure routes of maduramicin in the abdominal muscle and the hepatopancreas tissue homogenates of crayfish were given intragastrically to mice in different doses for seven days. Action changes, clinical symptoms, feed consumption, body weight, blood biochemistry, and histopathology examination of mice were observed and analyzed. In the natural exposure group, relatively low concentration of maduramicin in crayfish muscle and hepatopancreas had no obvious effects on mental state, body weight, blood biochemical indexes, or histologic appearance. However, in the artificial exposure group, with increasing concentrations, maduramicin in crayfish muscle and hepatopancreas homogenates both induced mental sluggishness and weight loss of mice. Blood biochemical examination showed that 3.5 mg·kg-1 and 7 mg·kg-1 maduramicin in crayfish tissue homogenates significantly increased levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and creatine kinase (CK). Additionally, histopathological examination showed that multiple organs were damaged by maduramicin, including degeneration of liver cells, shedding of renal epithelial cells, and disturbance and partial lysis of myocardial and skeletal muscle filaments in the mice. In summary, maduramicin may not cause Haff disease through contamination of the aquatic environment under normal conditions. Maduramicin can be used as a potential toxin tool to establish a rhabdomyolysis disease animal model for drug development.

Maduramicin triggers methuosis-like cell death in primary chicken myocardial cells.

Maduramicin frequently induces severe cardiotoxicity in broiler chickens as well as in humans who consume maduramicin accidentally. Apoptosis and non-apoptotic cell death occur concurrently in the process of maduramicin-induced cardiotoxicity; however, the underlying mechanism of non-apoptotic cell death is largely unknown. Here, we report the relationship between maduramicin-caused cytoplasmic vacuolization and methuosis-like cell death as well as the underlying mechanism in primary chicken myocardial cells. Maduramicin induced a significant increase of cytoplasmic vacuoles with a degree of cell specificity in primary chicken embryo fibroblasts and chicken hepatoma cells (LMH), along with a decrease of ATP and an increase of LDH. The accumulated vacuoles were partly derived from cellular endocytosis rather than the swelling of endoplasm reticulum, lysosomes, and mitochondria. Moreover, the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) did not prevent maduramicin-induced cytoplasmic vacuolization. DNA ladder and cleavage of PARP were not observed in chicken myocardial cells during maduramicin exposure. Pretreatment with 3-methyladenine (3-MA) and cholorquine (CQ) of chicken myocardial cells did not attenuate cytoplasmic vacuolization and cytotoxicity, although LC3 and p62 were activated. Bafilomycin A1 almost completely prevented the generation of cytoplasmic vacuoles and significantly attenuated cytotoxicity induced by maduramicin, along with downregulation of K-Ras and upregulation of Rac1. Taken together, "methuosis" due to excessive cytoplasmic vacuolization mediates the cardiotoxicity of maduramicin. This provides new insights for understanding a nonclassical form of cell death in the field of drug-induced cytotoxicity.

Antitumor effects of anlotinib in thyroid cancer.

There is no effective treatment for patients with poorly differentiated papillary thyroid cancer or anaplastic thyroid cancer (ATC). Anlotinib, a multi-kinase inhibitor, has already shown antitumor effects in various types of carcinoma in a phase I clinical trial. In this study, we aimed to better understand the effect and efficacy of anlotinib against thyroid carcinoma cells in vitro and in vivo. We found that anlotinib inhibits the cell viability of papillary thyroid cancer and ATC cell lines, likely due to abnormal spindle assembly, G2/M arrest, and activation of TP53 upon anlotinib treatment. Moreover, anlotinib suppresses the migration of thyroid cancer cells in vitro and the growth of xenograft thyroid tumors in mice. Our data demonstrate that anlotinib has significant anticancer activity in thyroid cancer, and potentially offers an effective therapeutic strategy for patients of advanced thyroid cancer type.

A high-throughput method for screening of L-tyrosine high-yield strains by Saccharomyces cerevisiae.

A biosensor screening assay based on the synthesis of betaxanthin was applied to relatively high throughput screening of the L-tyrosine mutant library. In the assays, fluorescence output showed a linear relationship between extracellular L-tyrosine content and yellow pigment formation. In addition, the yellow pigment accumulation of the L-tyrosine high-yield strain can be easily distinguished with the naked eye compared with the wild-type strain. As a result, numerous mutants that exhibited significantly increased coloration, were screened out after random mutagenesis, and p-coumaric acid production in mutants NK-A3 and NK-B4, were remarkably improved by 4-fold more than that of the wild-type strain. In general, this study provides a novel strategy for screening mutant libraries in the search for highly L-tyrosine-producing strains.