A Distinctive Insight into Inorganic Sonosensitizers: Design Principles and Application Domains.

Sonodynamic therapy (SDT) as a promising non-invasive anti-tumor means features the preferable penetration depth, which nevertheless, usually can't work without sonosensitizers. Sonosensitizers produce reactive oxygen species (ROS) in the presence of ultrasound to directly kill tumor cells, and concurrently activate anti-tumor immunity especially after integration with tumor microenvironment (TME)-engineered nanobiotechnologies and combined therapy. Current sonosensitizers are classified into organic and inorganic ones, and current most reviews only cover organic sonosensitizers and highlighted their anti-tumor applications. However, there have few specific reviews that focus on inorganic sonosensitizers including their design principles, microenvironment regulation, etc. In this review, inorganic sonosensitizers are first classified according to their design rationales rather than composition, and the action rationales and underlying chemistry features are highlighted. Afterward, what and how TME is regulated based on the inorganic sonosensitizers-based SDT nanoplatform with an emphasis on the TME targets-engineered nanobiotechnologies are elucidated. Additionally, the combined therapy and their applications in non-cancer diseases are also outlined. Finally, the setbacks and challenges, and proposed the potential solutions and future directions is pointed out. This review provides a comprehensive and detailed horizon on inorganic sonosensitizers, and will arouse more attentions on SDT.

Vertical sleeve gastrectomy confers metabolic improvements by reducing intestinal bile acids and lipid absorption in mice.

Vertical sleeve gastrectomy (VSG) is one of the most effective and durable therapies for morbid obesity and its related complications. Although bile acids (BAs) have been implicated as downstream mediators of VSG, the specific mechanisms through which BA changes contribute to the metabolic effects of VSG remain poorly understood. Here, we confirm that high fat diet-fed global farnesoid X receptor (Fxr) knockout mice are resistant to the beneficial metabolic effects of VSG. However, the beneficial effects of VSG were retained in high fat diet-fed intestine- or liver-specific Fxr knockouts, and VSG did not result in Fxr activation in the liver or intestine of control mice. Instead, VSG decreased expression of positive hepatic Fxr target genes, including the bile salt export pump (Bsep) that delivers BAs to the biliary pathway. This reduced small intestine BA levels in mice, leading to lower intestinal fat absorption. These findings were verified in sterol 27-hydroxylase (Cyp27a1) knockout mice, which exhibited low intestinal BAs and fat absorption and did not show metabolic improvements following VSG. In addition, restoring small intestinal BA levels by dietary supplementation with taurocholic acid (TCA) partially blocked the beneficial effects of VSG. Altogether, these findings suggest that reductions in intestinal BAs and lipid absorption contribute to the metabolic benefits of VSG.

Effects of Flavourzyme and Alkaline Protease Treatment on Structure and Allergenicity of Peanut Allergen Ara h 1.

Research background: Peanut allergy poses a significant threat to human health due to the increased risk of long-term morbidity at low doses. Modifying protein structure to affect sensitization is a popular topic. Experimental approach: In this study, the purified peanut allergen Ara h 1 was enzymatically hydrolysed using Flavourzyme, alkaline protease or a combination of both. The binding ability of Ara h 1 to antibodies, gene expression and secretion levels of the proinflammatory factors interleukin-5 and interleukin-6 in Caco-2 cells was measured. Changes in the secondary and tertiary structures before and after treatment with Ara h 1 were analysed by circular dichroism and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results and conclusions: The results indicated a decrease of the allergenicity and proinflammatory ability of Ara h 1. The evaluation showed that the Flavourzyme and alkaline protease treatments caused particle shortening and aggregation. The fluorescence emission peak increased by 3.4-fold after the combined treatment with both proteases. Additionally, the secondary structure underwent changes and the hydrophobicity also increased 8.95-fold after the combined treatment. Novelty and scientific contribution: These findings partially uncover the mechanism of peanut sensitization and provide an effective theoretical basis for the development of a new method of peanut desensitization.

Drug-induced liver injury following the use of tocilizumab or sarilumab in patients with coronavirus disease 2019.

BACKGROUNDS: Interleukin-6 (IL-6) blockers including tocilizumab and sarilumab were approved by the U.S. Food and Drug Administration (FDA) in June 2021 for the treatment of patients with moderate to severe COVID-19. The use of sarilumab or tocilizumab in COVID-19 patients has been related to a reduction in mortality compared to standard care. Recent evidence has emerged concerning drug-induced liver injury (DILI) after sarilumab or tocilizumab applications in COVID-19 patients. AIMS: The study aimed to estimate DILI associated with sarilumab or tocilizumab in treating moderate to severe patients infected with SARS-Cov-2. METHODS: We conducted a retrospective pharmacovigilance study by data mining of the FDA's adverse event reporting systems (FAERS) database from the first quarter of 2004 to the fourth quarter of 2021 in confirmed COVID-19 patients. We analyzed DILI cases associated with tocilizumab or sarilumab in treating COVID-19 patients from the FAERS during this period. Disproportionality analysis and Bayesian analysis of COVID-19 patients were utilized for case analysis, and we also next compared the onset time and fatality rates of DILI following tocilizumab or sarilumab. RESULTS: A total of 275 cases of TCZ or SAR-related DILI reports were extracted. A total of 192 AEs cases were related to tocilizumab (TCZ), and 83 were related to sarilumab (SAR). In patients treated with TCZ, most were < 75 years old (51.57%), with more male than female (46.35% vs. 13.02%). The correlation between IL-6 receptor antagonists and DILI was stronger in SAR (ROR = 12.94; 95%CI 9.6-17.44) than in TCZ (ROR = 1.33; 95%CI 1.14-1.55). The onset time of DILI was different between TCZ and SAR, and a significant difference was observed in TCZ than SAR (P < 0.0001). A significant difference was observed in the mortality rate of TCZ and SAR (P = 0.0009). DILI associated with COVID-19 patients treated with TCZ appeared to have earlier onset-time (1(0-46) day) VS. SAR (3.5(0-27) day). CONCLUSION: This study shows strict monitor ought to be paid for TCZ or SAR when used for COVID-19 patients with poor liver function.

Epidemiologic and import risk analysis of Peste des petits ruminants between 2010 and 2018 in India.

BACKGROUND: Peste des petits ruminants (PPR) is a serious disease that affects goats, sheep and other small ruminants. As one of the earliest and most serious countries, PPR has seriously threatened India's animal husbandry economy. RESULTS: In this study, the spatiotemporal characteristics of the PPR in India outbreaks were analyzed. Between 2010 and 2018, the epidemic in India broke out all over the country in a cluster distribution. Epidemic clusters in northern and southern India are at higher risk, and the outbreak time of PPR has significant seasonality. The results of the analysis of the development and transmission of PPR under the natural infection conditions showed that the PPR outbreak in India reached a peak within 15 days. Finally, the quantitative risk analysis results based on scenario tree show showed that the average probability of infecting PPRV in live sheep exported from India was 1.45 × 10-4. CONCLUSIONS: This study analyzed the prevalence of PPR in India. The analysis of transmission dynamics on the development of the epidemic provides a reference for the prevention and control of the epidemic. At the same time, it provides risk analysis and suggestions on trade measures for the trading countries of India.

An Improved POD Model for Fast Semi-Quantitative Analysis of Carbendazim in Fruit by Surface Enhanced Raman Spectroscopy.

The current detection method of carbendazim suffers from the disadvantages of complicated preprocessing and long cycle time. In order to solve the problem of rapid quantitative screening of finite contaminants, this article proposed a qualitative method based on characteristic peaks and a semi-quantitative method based on threshold to detect carbendazim in apple, and finally the method is evaluated by a validation system based on binary output. The results showed that the detection limit for carbendazim was 0.5 mg/kg, and the detection probability was 100% when the concentration was no less than 1 mg/kg. The semi-quantitative analysis method had a false positive rate of 0% and 5% at 0.5 mg/kg and 2.5 mg/kg, respectively. The results of method evaluation showed that when the added concentration was greater than 2.5 mg/kg, the qualitative detection method was consistent with the reference method. When the concentration was no less than 5 mg/kg, the semi-quantitative method is consistent between different labs. The semi-quantitative method proposed in this study can achieve the screening of finite contaminants in blind samples and simplify the test validation process through the detection probability model, which can meet the needs of rapid on-site detection and has a good application prospect.

Screening of potential vaccine candidates against pathogenic Brucella spp. using compositive reverse vaccinology.

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and various animals. The threat of brucellosis has increased, yet currently available live attenuated vaccines still have drawbacks. Therefore, subunit vaccines, produced using protein antigens and having the advantage of being safe, cost-effective and efficacious, are urgently needed. In this study, we used core proteome analysis and a compositive RV methodology to screen potential broad-spectrum antigens against 213 pathogenic strains of Brucella spp. with worldwide geographic distribution. Candidate proteins were scored according to six biological features: subcellular localization, antigen similarity, antigenicity, mature epitope density, virulence, and adhesion probability. In the RV analysis, a total 32 candidate antigens were picked out. Of these, three proteins were selected for assessment of immunogenicity and preliminary protection in a mouse model: outer membrane protein Omp19 (used as a positive control), type IV secretion system (T4SS) protein VirB8, and type I secretion system (T1SS) protein HlyD. These three antigens with a high degree of conservation could induce specific humoral and cellular immune responses. Omp19, VirB8 and HlyD could substantially reduce the organ bacterial load of B. abortus S19 in mice and provide varying degrees of protection. In this study, we demonstrated the effectiveness of this unique strategy for the screening of potential broad-spectrum antigens against Brucella. Further evaluation is needed to identify the levels of protection conferred by the vaccine antigens against wild-type pathogenic Brucella species challenge.

Development and validation of a rapid and sensitive LC-MS/MS method for the determination of polyphyllin II in rat plasma and its application in a pharmacokinetic study.

Polyphyllin II, a major steroidal saponin isolated from Paris polyphylla, exhibits significant pharmacological activities. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was established and validated for the determination of polyphyllin II in plasma. Polyphyllin II and polyphyllin VII (internal standard) were separated on a Waters Acquity™ HSS T3 column and the mass analysis was performed in a triple quadrupole mass spectrometer equipped with an electrospray ionization ion source. Results showed that the method was sensitive (lower limit of quantitation 0.5 ng/ml), precise (<15%) and linear in the range of 0.5-500 ng/ml (r > 0.99). Interestingly, the sensitivity in current study was ~10 times higher than that in the previous study. The results of the pharmacokinetic study of polyphyllin II in rats suggested that polyphyllin II was poorly absorbed into blood and reached its highest concentration at ~3.67-5.00 h with a slow elimination half-life of 8.34-13.37 h. The bioavailability was 6.1-8.2%. The results indicated that the absorption of polyphyllin II may primarily occur via passive diffusion in rats. This study provides valuable information that can be used as a reference for the pharmacokinetic investigation of other steroidal saponins.

Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality.

Adenoviral vectors (AdV) are considered promising candidates for vaccine applications. A prominent group of Toll-like receptors (TLRs) participate in the adenovirus-induced adaptive immune response, yet there is little information regarding the role of TLR4 in AdV-induced immune responses in recent literature. We investigated the function of TLR4 in both adaptive and innate immune responses to an AdV-based anthrax vaccine. By immunizing wild-type and TLR4 knockout (TLR4-KO) mice, we revealed the requirement of TLR4 in AdV-induced innate responses. We also showed that TLR4 functions are required for germinal centre responses in immunized mice, as expression of the apoptosis-related marker Fas was down-regulated on germinal centre B cells from TLR4-KO mice. Likewise, decreased expression of inducible costimulator on follicular T helper cells was observed in immunized TLR4-KO mice. Moreover, a potent protective antigen-specific humoral immune response was mimicked using an adjuvant system containing the TLR4 agonist monophosphoryl lipid A. Overall, our findings showed that very rapid antigen-specific antibody production is correlated with the TLR4-imprinted germinal centre response to AdV-based vaccine. These results provide additional evidence for the use of the AdV and a TLR agonist to induce humoral responses. Our findings offer new insights into rational vaccine design.

A comprehensive proteogenomic study of the human Brucella vaccine strain 104 M.

BACKGROUND: Brucella spp. are Gram-negative, facultative intracellular pathogens that cause brucellosis in both humans and animals. The B. abortus vaccine strain 104 M is the only vaccine available in China for the prevention of brucellosis in humans. Although the B. abortus 104 M genome has been fully sequenced, the current genome annotations are not yet complete. In addition, the main mechanisms underpinning its residual toxicity and vaccine-induced immune protection have yet to be elucidated. Mapping the proteome of B. abortus 104 M will help to improve genome annotation quality, thereby facilitating a greater understanding of its biology. RESULTS: In this study, we utilized a proteogenomic approach that combined subcellular fractionation and peptide fractionation to perform a whole-proteome analysis and genome reannotation of B. abortus 104 M using high-resolution mass spectrometry. In total, 1,729 proteins (56.3% of 3,072) including 218 hypothetical proteins were identified using the culture conditions that were employed this study. The annotations of the B. abortus 104 M genome were also refined following identification and validation by reverse transcription-PCR. In addition, 14 pivotal virulence factors and 17 known protective antigens known to be involved in residual toxicity and immune protection were confirmed at the protein level following induction by the 104 M vaccine. Moreover, a further insight into the cell biology of multichromosomal bacteria was obtained following the elucidation of differences in protein expression levels between the small and large chromosomes. CONCLUSIONS: The work presented in this report used a proteogenomic approach to perform whole-proteome analysis and genome reannotation in B. abortus 104 M; this work helped to improve genome annotation quality. Our analysis of virulence factors, protective antigens and other protein effectors provided the basis for further research to elucidate the mechanisms of residual toxicity and immune protection induced by the 104 M vaccine. Finally, the potential link between replication dynamics, gene function, and protein expression levels in this multichromosomal bacterium was detailed.

Vertical sleeve gastrectomy activates GPBAR-1/TGR5 to sustain weight loss, improve fatty liver, and remit insulin resistance in mice.

UNLABELLED: Vertical sleeve gastrectomy (VSG) is one of the most commonly performed clinical bariatric surgeries used for the remission of obesity and diabetes. However, the precise molecular mechanism by which VSG exerts its beneficial effects remains elusive. We report that the membrane-bound G protein-coupled bile acid receptor, GPBAR-1 (also known as TGR5), is required to mediate the effects of anti-obesity, anti-hyperglycemia, and improvements of fatty liver of VSG in mice. In the absence of TGR5, the beneficial metabolic effects of VSG in mice are lost. Moreover, we found that the expression of TGR5 increased significantly after VSG, and VSG alters both BA levels and composition in mice, resulting in enhancement of TGR5 signaling in the ileum and brown adipose tissues, concomitant with improved glucose control and increased energy expenditure. CONCLUSION: Our study elucidates a novel underlying mechanism by which VSG achieves its postoperative therapeutic effects through enhanced TGR5 signaling. (Hepatology 2016;64:760-773).

Sweroside ameliorates α-naphthylisothiocyanate-induced cholestatic liver injury in mice by regulating bile acids and suppressing pro-inflammatory responses.

AIM: Sweroside is an iridoid glycoside with diverse biological activities. In the present study we investigated the effects of sweroside on α-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury in mice. METHODS: Mice received sweroside (120 mg·kg(-1)·d(-1), ig) or a positive control INT-747 (12 mg·kg(-1)·d(-1), ig) for 5 d, and ANIT (75 mg/kg, ig) was administered on d 3. The mice were euthanized on d 5, and serum biochemical markers, hepatic bile acids and histological changes were analyzed. Hepatic expression of genes related to pro-inflammatory mediators and bile acid metabolism was also assessed. Primary mouse hepatocytes were exposed to a reconstituted mixture of hepatic bile acids, which were markedly elevated in the ANIT-treated mice, and the cell viability and expression of genes related to pro-inflammatory mediators were examined. RESULTS: Administration of sweroside or INT-747 effectively ameliorated ANIT-induced cholestatic liver injury in mice, as evidenced by significantly reduced serum biochemical markers and attenuated pathological changes in liver tissues. Furthermore, administration of sweroside or INT-747 significantly decreased ANIT-induced elevation of individual hepatic bile acids, such as ß-MCA, CA, and TCA, which were related to its effects on the expression of genes responsible for bile acid synthesis and transport as well as pro-inflammatory responses. Treatment of mouse hepatocytes with the reconstituted bile acid mixture induced significant pro-inflammatory responses without affecting the cell viability. CONCLUSION: Sweroside attenuates ANIT-induced cholestatic liver injury in mice by restoring bile acid synthesis and transport to their normal levels, as well as suppressing pro-inflammatory responses.

Preparation and application of a coated-fiber needle extraction device.

In this study, a needle-trap device with fibers coated with a molecularly imprinted polymer was developed for separation. A number of heat-resistant Zylon filaments were longitudinally packed into a glass capillary, followed by coating with a molecularly imprinted polymer. Then, the molecularly imprinted polymer coating was copolymerized and anchored onto the surface of the fibers. The bundle of synthetic fibers coated with the molecularly imprinted polymer was packed into a 21G stainless-steel needle and served as an extraction medium. The coated-fiber needle extraction device was used to extract volatile organic compounds from paints and gasoline effectively. Subsequently, the extracted volatile organic compounds were analyzed by gas chromatography. Calibration curves of gaseous benzene, toluene, ethylbenzene, and o-xylene in the concentration range of 1-250 µg/L were obtained to evaluate the method, acceptable linearity was attended with correlation coefficients above 0.998. The limit of detection of benzene, toluene, ethylbenzene, and o-xylene was 11-20 ng/L using the coated-fiber needle-trap device. The relative standard deviation of needle-to-needle repeatability was less than 8% with an extraction time of 20 min. The loss rates after storage for 3 and 7 days at room temperature were less than 30%.

Chlorpromazine-induced perturbations of bile acids and free fatty acids in cholestatic liver injury prevented by the Chinese herbal compound Yin-Chen-Hao-Tang.

BACKGROUNDS: Yin-Chen-Hao-Tang (YCHT), a commonly used as a traditional chinese medicine for liver disease. Several studies indicated that YCHT may improving hepatic triglyceride metabolism and anti-apoptotic response as well as decreasing oxidative stress .However, little is known about the role of YCHT in chlorpromazine (CPZ) -induced chlolestatic liver injury. Therefore, we aimed to facilitate the understanding of the pathogenesis of cholestatic liver injury and evaluate the effect of Yin-Chen-Hao-Tang (YCHT) on chlorpromazine (CPZ)-induced cholestatic liver injury in rats based on the change of bile acids (BAs) and free fatty acids (FFAs) alone with the biochemical indicators and histological examination. METHODS: We conducted an experiment on CPZ-induced cholestatic liver injury in Wistar rats with and without YCHT for nine consecutive days. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin (TBIL), total cholesterol (TC), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C) were measured to evaluate the protective effect of YCHT against chlorpromazine (CPZ)-induced cholestatic liver injury. Histopathology of the liver tissue showed that pathological injuries were relieved after YCHT pretreatment. In addition, ultra-performance lipid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) and gas chromatography coupled with mass spectrometry (GC-MS) was applied to determine the content of bile acids, free fatty acids, respectively. RESULTS: Obtained data showed that YCHT attenuated the effect of CPZ-induced cholestatic liver injury, which was manifested by the serum biochemical parameters and histopathology of the liver tissue. YCHT regulated the lipid levels as indicated by the reversed serum levels of TC, TG, and LDL-C. YCHT also regulated the disorder of BA and FFA metabolism by CPZ induction. CONCLUSIONS: Results indicated that YCHT exerted a protective effect on CPZ-induced cholestasis liver injury. The variance of BA and FFA concentrations can be used to evaluate the cholestatic liver injury caused by CPZ and the hepatoprotective effect of YCHT.

Andrographolide prevents high-fat diet-induced obesity in C57BL/6 mice by suppressing the sterol regulatory element-binding protein pathway.

Sterol regulatory element-binding proteins (SREBPs) are major transcription factors regulating the expression of genes involved in biosynthesis of cholesterol, fatty acids, and triglycerides. We investigated the effect of the specific SREBP suppressor andrographolide, a natural compound isolated from Andrographis paniculata, on the regulation of SREBP signaling by use of Western blot, reporter gene assay, and quantitative real-time polymerase chain reaction analysis. In addition, the antiobesity effects of andrographolide were evaluated in C57BL/6 mice with high-fat diet (HFD)-induced obesity. Our results showed that andrographolide downregulated the expressions of SREBPs target genes and decreased cellular lipid accumulation in vitro. Further, andrographolide (100 mg/kg per day) attenuated HFD-induced body weight gain and fat accumulation in liver or adipose tissues, and improved serum lipid levels and insulin or glucose sensitivity in HFD-induced obese mice. Andrographolide effectively suppressed the respiratory quotient, energy expenditure, and oxygen consumption, which may have contributed to the decreased body-weight gain of the obese mice fed with a HFD. Consistently, andrographolide regulated SREBP target genes and metabolism-associated genes in liver or brown adipose tissue, which may have directly contributed to the lower lipid levels and enhanced insulin sensitivity. Taken together, our results indicated that andrographolide ameliorated lipid metabolism and improved glucose use in mice with HFD-induced obesity. Andrographolide has potential as a leading compound in the prevention or treatment of obesity and insulin resistance.

Leonurine alleviates vancomycin nephrotoxicity via activating PPARγ and inhibiting the TLR4/NF-κB/TNF-α pathway.

Vancomycin (VCM) is the first-line antibiotic for severe infections, but nephrotoxicity limits its use. Leonurine (Leo) has shown protective effects against kidney damage. However, the effect and mechanism of Leo on VCM nephrotoxicity remain unclear. In this study, mice and HK-2 cells exposed to VCM were treated with Leo. Biochemical and pathological analysis and fluorescence probe methods were performed to examine the role of Leo in VCM nephrotoxicity. Immunohistochemistry, q-PCR, western blot, FACS, and Autodock software were used to verify the mechanism. The present results indicate that Leo significantly alleviates VCM-induced renal injury, morphological damage, and oxidative stress. Increased intracellular and mitochondrial ROS in HK-2 cells and decreased mitochondrial numbers in mouse renal tubular epithelial cells were reversed in Leo-administrated groups. In addition, molecular docking analysis using Autodock software revealed that Leo binds to the PPARγ protein with high affinity. Mechanistic exploration indicated that Leo inhibited VCM nephrotoxicity via activating PPARγ and inhibiting the TLR4/NF-κB/TNF-α inflammation pathway. Taken together, our results indicate that the PPARγ inhibition and inflammation reactions were implicated in the VCM nephrotoxicity and provide a promising therapeutic strategy for renal injury.

Sonocatalytic oncolysis microbiota curb intrinsic microbiota lactate metabolism and blockade CD24-Siglec10 immune escape to revitalize immunological surveillance.

Intrinsic lactate retention of chemically- or genetically-engineered bacteria therapy aggravates tumor immunosuppression, which will collaborate with immune escape to cause immunological surveillance failure. To address them, sonocatalytic oncolysis Escherichia coli (E.coli) that chemically chelated anti-CD24 and TiO1+x have been engineered to blockade CD24-siglec10 interaction, regulate microbiota colonization and curb its lactate metabolism, which are leveraged to revitalize immunological surveillance and repress breast cancer. The chemically-engineered E.coli inherited their parent genetic information and expansion function. Therefore, their intrinsic hypoxia tropism and CD24 targeting allow them to specifically accumulate and colonize in solid breast cancer to lyse tumor cells. The conjugated CD24 antibody is allowed to blockade CD24-Siglec10 signaling axis and revitalize immunological surveillance. More significantly, the chelated TiO1+x sonosensitizers produce ROS to render bacteria expansion controllable and curb immunosuppression-associated lactate birth that are usually neglected. Systematic experiments successfully vlaidate hypoxia-objective active targeting, sonocatalytic therapy, microbiota expansion-enabled oncolysis, CD24-Siglec10 communication blockade and precise microbiota abundance & lactate metabolism attenuations. These actions contribute to the potentiated anti-tumor immunity and activated anti-metastasis immune memory against breast cancer development. Our pioneering work provide a route to sonocatalytic cancer immunotherapy.

Treatment with gentamicin on a murine model of protothecal mastitis.

The aim of this study was to establish a murine protothecal mastitis model and to evaluate the treatment efficiency of gentamicin. Challenge routes were determined with a pathogenic Prototheca zopfii genotype 2 (P. zopfii) strain. 25 BALB/c mice were inoculated in mammary glands with graded dosages (10(3), 10(4), 10(5), 10(6), 10(7) CFU of P. zopfii) and killed on the 7th day. Another 25 animals were also killed at 1, 3, 5, 7, and 9 days after inoculation of 1 × 10(6) CFU of P. zopfii, the milk somatic cell counts, pathological section of mammary glands, and P. zopfii burden were observed. The antimicrobial activity was tested using disc diffusion test and minimum inhibitory concentrations. Gentamicin was given intramuscularly to analyze the therapeutic effect. The results showed that the best infection route was intra-mammary gland, and the mastitis model was established with 1 × 10(6) CFU of P. zopfii. After infection, the somatic cell counts increased significantly. The pathological reaction mainly consisted of infiltration of inflammatory cells, destruction of acini, accumulation of lymphocyte cells and the severity of the changes was dosage and time-dependent. The P. zopfii burden revealed that P. zopfii continuously replicated. In vitro susceptibility tests indicated that the Prototheca strains were antimicrobial susceptible to gentamicin at concentrations between 0.03 and 4 µg/ml. In vivo therapeutic assay demonstrated that high concentrations of gentamicin (≥20 mg/kg) could inhibit the growth of P. zopfii. We conclude that the murine model of protothecal mastitis was established successfully and gentamicin may be an effective choice for treatment of P. zopfii.

Vancomycin Induced Ferroptosis in Renal Injury Through the Inactivation of Recombinant Glutathione Peroxidase 4 and the Accumulation of Peroxides.

Background: Vancomycin (VCM) has long been used clinically to fight against Gram-positive bacterial infections. In recent decades, an increased number of kidney injury cases caused by VCM overdose have been reported. In this study, we further investigated the mechanism of VCM-overdose-induced kidney injury. Methods: Immunohistochemistry (IHC) staining, RT-qPCR and Western blot assays were used to determine ki67, DDX5, PTGS2, GPX4 and SLC7A11 expressions in the kidney tissues of mice. CCK-8 and flow cytometry assays were used to determine HK2 cell viability and apoptosis. In addition, RT-qPCR and Western blot assays was applied to evaluate the expressions of ACSL4, PTGS2, GPX4, SLC7A11, DDX5 and Ki67 in HK2 cells. Results: We found that VCM induced ferroptosis in vitro and in vivo. Ferrostatin-1 (Fer-1) is a potent inhibitor of ferroptosis, Fer-1 rescued cell viability and renal function renal morphology in VCM-treated cells and mice, respectively. Further, GPX4, which plays an essential role in reducing lipid hydroperoxides and preventing ferroptosis, was observed to be downregulated by VCM treatment. Interestingly, we found that GPX4-knockdown HK-2 cells exhibited a similar phenotype and gene expression level of ACSL4, PTGS2, DDX5 and Ki67 compared with VCM-treated cells, which suggested that VCM could induce ferroptosis in HK2 cells by down-regulating GPX4. Conclusion: In conclusion, VCM induced renal injury in the kidney tissues of mice. In addition, VCM induced ferroptosis cell death in HK-2 cells and in the kidney tissues of mice by down-regulating GPX4 and causing the accumulation of peroxides. These data suggested that VCM could induce renal injury in vitro and in vivo via triggering ferroptosis. This study further elucidates the mechanism of VCM-induced renal injury and provides additional references for clinical use of VCM.