Multidrug-resistant (MDR) pathogens pose a serious threat to the health and life of humans, necessitating the development of new antimicrobial agents. Herein, we develop and characterize a panel of nine amino acid peptides with a cation end motif. Bioactivity analysis revealed that the short peptide containing "RWWWR" as a central motif harboring mirror structure "KXR" unit displayed not only high activity against MDR planktonic bacteria but also a clearance rate of 92.33% ± 0.58% against mature biofilm. Mechanically, the target peptide (KLR) killed pathogens by excessively accumulating reactive oxygen species and physically disrupting membranes, thereby enhancing its robustness for controlling drug resistance. In the animal model of sepsis infection by MDR bacteria, the peptide KLR exhibited strong therapeutic effects. Collectively, this study provided the dominant structure of short antimicrobial peptides (AMPs) to replenish our arsenals for combating bacterial infections and illustrated what could be harnessed as a new agent for fighting MDR bacteria.

Abstract In this study Ethanamizuril in different dispersants were administrated orally to target animal, chickens for pharmacokinetic study. An ultra-performance liquid chromatography method was established for sample detection and the extraction method was validated. Internal standard was used to calculate the drug concentration in chicken plasma. The results showed that the peak time of Ethanamizuril in chicken under the three dispersants was 4 ~ 5 hours, and the half-life (t1/2) of Ethanamizuril in sodium carboxymethyl cellulose solution (CMC-Na), PEG300 and Ethanamizuril solution diluted by water had a significant difference (P < 0.05) and AUC and Cmax had no significant difference which also showed no significant difference in drug exposure. AUC and Cmax were important indicators of drug exposure in toxicity studies. The study showed that different dispersants had little effect on the evaluation of toxicity studies of the drug.

Chronicwound infection caused by multidrug-resistant bacteriais a major threat globally, leading to high mortality rates and aconsiderable economic burden. To address it, an innovative supramolecularnanofiber hydrogel (Hydrogel-RL) harboring antimicrobial peptideswas developed based on the novel arginine end-tagging peptide (Pep6) from our recent study, triggering cross-linking. In vitro resultsdemonstrated that Hydrogel-RL can sustain the release of Pep 6 upto 120 h profiles, which is biocompatible and exhibits superior activityfor methicillin-resistant Staphylococcus aureus (MRSA) biofilm inhibition and elimination. A single treatment ofsupramolecular Hydrogel-RL on an MRSA skin infection model revealedformidable antimicrobial activity and therapeutic effects in vivo.In the chronic wound infection model, Hydrogel-RL promoted mouse skincell proliferation, reduced inflammation, accelerated re-epithelialization,and regulated muscle and collagen fiber formation, rapidly healingfull-thickness skin wounds. To show its vehicle property for woundinfection combined therapy, etamsylate, an antihemorrhagic drug, wasloaded into the porous network of Hydrogel-RL, which demonstratedimproved hemostatic activity. Collectively, Hydrogel-RL is a promisingclinical candidate agent for functional supramolecular biomaterialsdesigned for combating multidrug-resistant bacteria and rescuing stalledhealing in chronic wound infections.

Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.

The green fluorescent protein (GFP)-based reporter system has been widely harnessed as a quick quantitative activity assessment method for characterizing CRISPR-Cas via flow cytometry. However, due to the small size (738 nt) of the GFP coding sequence, the targeting sites for certain CRISPR-Cas are greatly restricted. To address this, here we developed a GFP tagged polycistronic reporter system to determine the activity of CRISPR-Cas in human cells. Specifically, the system contains the herpes simplex virus thymidine kinase (TK) gene, bacterial neomycin phosphotransferase (Neo) gene, and green fluorescent protein (GFP), named TNG gene, with a coding sequence of 2,577 nt. To investigate its performance, we generated a human cell line harboring the TNG expression cassette at the AAVS1 locus, and then we tested it with different Cas orthologs (SaCas9, St1Cas9, and AsCas12a). Our results demonstrated that using the TNG reporter system greatly expands the targeting site selection (3- to 13-fold) with CRISPR-Cas genome editing. The study therefore reports an additional method for the characterization of CRISPR-Cas technology.

Chicken coccidiosis is one of the most common and economically important diseases in the global poultry industry, and it is caused by at least one of the seven Eimeria species. A simple and reliable way to distinguish Eimeria species in infected chicken is critical for the surveillance, control, and eradication of chicken coccidiosis. In this study, a recombinase polymerase amplification (RPA) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system (RPA-CRISPR/Cas12a) was developed for the detection of Eimeria species in chicken fecal samples. This assay is highly specific to the seven Eimeria species and it does not cross react between species. Assessment of analytical sensitivity revealed that a single copy of plasmid DNA could be detected. Comparative analysis revealed strong agreement between RPA-CRISPR/Cas12a assays and real-time qPCR to reliably detect all seven Eimeria species in fecal chicken samples. Importantly, the cleavage products could be visualized under a blue light instrument, making it possible for the rapid detection of Eimeria species for on-site testing. Collectively, our study demonstrated that RPA-CRISPR/Cas12a assays offer a simple and reliable diagnostic method for Eimeria species.

沙咪珠利(EZL)是中国农业科学院上海兽医研究所自主研发的一种新型三嗪类抗球虫药物.本研究通过建立鸡球虫感染模型,进行感染鸡灌胃给药沙咪珠利4 mg/kg.bw与8 mg/kg.bw两种剂量后的药动学研究,采用高效液相色谱法测定鸡血浆中沙咪珠利药物浓度,使用DAS3.0非房室模型拟合分析药动学参数.选取健康鸡160只于15日龄时每只鸡感染柔嫩艾美尔球虫孢子化卵囊80000,感染后鸡只分为两组,每组分别选取75只感染模型于第5 d分别灌胃给药沙咪珠利4 mg/kg.bw与8 mg/kg.bw.感染鸡给药前后分别于0、0.5、1、2、3、4、6、8、12、24、36、48、60、72 h,每个时间点取5只鸡心脏采血,血浆样品-20℃保存待测.结果表明,感染鸡灌胃给药4 mg/kg.bw与8 mg/kg.bw后其最高血药浓度(Cmax)分别为4.28±1.56μg/mL与11.82±0.59μg/mL;达峰时间(Tmax)分别为5.2±3.83 h与8.8±1.80 h;AUC(0-t)分别96.90±16.31 mg/L*h与351.61±36.06 mg/L*h;消除半衰期(t1/2z)分别为13.83±5.13与15.07±7.47 h.

Background Avian coccidiosis is an important parasitic disease that has serious adverse effects on the global poultry industry. The extensive use of anticoccidial drugs has resulted in an increase in drug resistance. Ethanamizuril (EZL) is a novel triazine with high anticoccidial activity. Methods We compared oocyst production and sporulation between EZL-sensitive (S) and EZL-resistant Eimeria tenella strains (R10 and R200) and used label-free quantitative proteomics to identify differentially expressed proteins (DEPs) between these strains. Results We generated two EZL-resistant E. tenella strains: strain R10, which was induced using a constant dose of 10 mg EZL/kg poultry feed, and strain R200, which was generated by gradually increasing the EZL dosage to 200 mg EZL/kg poultry feed. With an increase in resistance, the total oocyst output decreased, but the percentage of sporulation did not change significantly. We identified a total of 7511 peptides and 1282 proteins, and found 152 DEPs in the R10 strain versus the S strain, 426 DEPs in the R200 strain versus the S strain and 494 DEPs in the R200 strain versus the R10 strain. When compared with the S strain, 86 DEPs were found to have consistent trends in both resistant strains. The DEPs were primarily involved in ATP and GTP binding, invasion, and membrane components. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the DEPs suggested that they are involved in transcription and translation processes. Protein–protein interaction network analysis of the 86 DEPs showed that 10 proteins were hubs in the functional interaction network (≥ 8 edges) and five of them were ribosomal proteins. Conclusions The results of the present study indicate that the resistance mechanisms of E. tenella against EZL might be related to the transcriptional and translational processes, especially in the factors that inhibit the growth of parasites. The DEPs found in this study provide new insights into the resistance mechanisms of E. tenella against EZL. Further research on these potential targets holds promise for new chemotherapeutic approaches for controlling E. tenella infections. Graphical Abstract

Eimeria species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella ( E. tenella ). Ectopic expression of Cas9, i.e., via plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as “FnCpf1”) protein and crRNA targeting EtHistone H4 triggered DNA double-strand breaks in vivo . We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein ( EYFP ) and dihydrofolate reductase–thymidylate synthase gene ( DHFR ) as a selection marker to tag endogenous EtActin . The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella , which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

本研究建立了超高效液相-串联质谱法(UPLC-MS/MS)检测鹅肌肉、肝脏、肾脏、皮脂组织中维吉尼亚霉素M1的残留检测方法.组织样品使用乙腈提取、正己烷脱脂,经过滤后直接运用超高效液相色谱-串联质谱检测,外标法定量,采用电喷雾离子源(ESI+),选择多反应监测(MRM)模式进行分析.结果表明:鹅各组织的定量限分别为肌肉10.0μg/kg、肝脏50.0μg/kg、肾脏50.0μg/kg、皮脂50.0μg/kg,平均回收率分别为肌肉93.09％～98.62％、肝脏98.41％～103.8％、肾脏101.5％～113.3％、皮脂100.9％～114.3％.批内、批间的RSDs均符合相关要求.该方法简化了样品前处理过程,具有较好的灵敏度、准确性,可用于检测鹅可食用组织中维吉尼亚霉素M1的残留量.

The treatment effect of ethanamizuril (EZL) to broiler chickens experimentally infected with 8 x 10(4) Eimeria tenella was evaluated. On the third day after infection, the broiler chickens were treated with EZL by gavage at doses of 2, 4, and 8 mg/kg body weight (bw) for once. For double administration, the challenged broiler chickens were administered EZL at doses of 1, 2, 4, and 8 mg/kg bw by gavage continually on the third day and fourth day and once a day. Throughout the experimental period, performance parameters including body weight gain, mortality, cecal lesion score, bloody diarrhoea and oocyst output were recorded. The anticoccidial efficacy was evaluated using the anticoccidial index (ACI). Meanwhile, the concentrations of EZL in chicken cecal contents were measured, and the data were analyzed with a non-compartmental model. The results indicated that EZL showed good anticoccidial activity at single dose of 4 mg/kgbw, with the corresponding ACI of 175.73. When the challenged chickens were treated with EZL under double administration, the EZL showed a medium level of anticoccidial activity at a dose of 2 mg/kg bw, with the corresponding ACI of 162.48. The maximum concentrations (C-max) of EZL in content were 2.43 +/- 1.16, 4.28 +/- 1.56, and 8.57 +/- 1.33 mg/kg after the chickens were administrated at doses of 2, 4, and 8 mg/kg bw, respectively. The respective areas under the curve were 36.93 +/- 8.91, 96 +/- 16.31, and 262.76 +/- 51.52 mg/kg h. The respective half-lives (T-1/(2)) were 10.82 +/- 2.02, 10.53 +/- 2.23, and 10.60 +/- 1.50 h. The results show that when the concentrations of EZL in chicken cecal contents reached 4.28 +/- 1.56 mg/kg, there is a significant therapeutic effect on chicken coccidiosis.

In the current study, to support the safety pharmacology assessment of Ethanamizuril as a new potent anticoccidial agent of triazine compounds, the effects of Ethanamizuril on the central nervous system, cardiovascular system and respiratory system were investigated. Using locomotor activity test, climbing behavior test and nembutal subthreshold hypnotic test at each time point after oral administration of Ethanamizuril to mice, the effects on the central nervous system were evaluated. An assessment of Ethanamizuril effects on the cardiovascular and respiratory system were performed by the use of a telemetry system in conscious beagle dogs. The results showed that the treatment of Ethanamizuril had no effects on motor activity, behavioral changes, coordination, and sensory/motor reflex responses in mice. There were also no changes in heart rate, blood pressure, and electrocardiogram at all doses and each time points in beagle dogs. Our data suggested that Ethanamizuril showed no adverse effects on the central nervous system, cardiovascular system, and respiratory system.

目的 建立高效液相法测定血浆中沙咪珠利的含量,并进行雏鸡口服给药后药代动力学研究.方法 试验采用高效液相法测定雏鸡血浆中的沙咪珠利,进行了方法学验证,包括专属性、线性、准确度及精密度、稳定性研究;给2 周龄雏鸡口服沙咪珠利,在各时间点从心脏采血,测定药物含量,采用DAS软件进行药代动力学计算.结果 采用高效液相测定鸡血浆中的沙咪珠利,血浆无干扰,内标分离度符合要求.0.05～20 μg/mL浓度范围线性良好,定量限为50 ng/mL,批间精密度均符合检测要求,相对回收率为100％～1 17％.雏鸡口服灌胃给药,Tmax 5.125 ±2.031 h,Cmax 9.964 ±0.895 μg/mL,t1/2为15.558 ±6.55 h;MRT为17.612 ±1.007 h.

BackgroundTriazine coccidiostats are widely used in chickens and turkeys for coccidiosis control. Ethanamizuril is a novel triazine compound that exhibits anticoccidial activity in poultry. This study was designed to evaluate the subchronic toxicity of ethanamizuril in beagle dogs at doses of 12, 60 or 300mg/kg/day in diet for 90days.ResultsEthanamizuril was well tolerated at low and middle dosages in beagle dogs, and no drug-related toxical effects were observaed in terms of survival, clinical observations, organs weight and damage in these dose groups. However, in high dose administration group, food consumption and histologic changes in kidneys were noticed in both sexes of beagle dog, although the renal lesions were finally resolved at the end of 4 weeks exposure of ethanamizuril.ConclusionsNo-observed-adverse-effect level (NOAEL) was considered for ethanamizuril at dose of 60mg/kg/day in Beagle dog. This result added toxicity effects of ethanamizuril to the safety database, which might guide safely using of ethanamizuril as a novel coccidiostat.

Ethanamizuril, a new anticoccidial agent that belongs to triazine derivatives, has a broad and good anticoccidial activity. To evaluate the reproductive toxicity and teratogenic potential of ethanamizuril, different concentrations of ethanamizuril were administered to Sprague-Dawley rats by feeding diets containing 0, 2, 8, and 30 ppm, respectively. Each group consisting of 30 males and 30 females (F0) was treated with different concentrations of ethanamizuril through a 13-week period before mating and during mating, gestation, parturition and lactation. In the 30 ppm dose group, pup body weight on days 7 and 21 in F1 offspring and day 21 in the F2a offspring were significantly decreased. A limited teratogenicity study was performed in combination with the F1 generation of a two-generation reproduction study. F1 offspring of the reproduction study were mated after weaning of the F2a offspring. Pregnant female rats were subjected to cesarean section on gestational day 20 for teratogenic examination. No obvious body weights, fetal body lengths, tail lengths, litter weights, number of viable fetuses, external, skeletal, or visceral malformations in fetuses were noted in any groups in the teratogenic test, but ethanamizuril could be passed on to offspring through lactation.

Ethanamizuril is a new triazine compound that shows potential for application in novel anticoccidial treatment. In this study, a pharmacokinetic model of ethanamizuril was established on the basis of the blood concentration of 81 experimental animals. The final model showed that ethanamizuril was distributed as a two-compartment model with first-order absorption after oral administration in chickens. Its clearance rate and volumn of central compartment distribution (Vc ) were affected by age and body weight, and volumn of central compartment distribution (Vc ) and volume of peripheral compartment distribution(Vp ) were influenced by weight and infection. External verification revealed that the model had good prediction accuracy and stability.

Ethanamizuril (EZL) is a novel triazine compound with excellent anticoccidial activity. We carried out a preliminary investigation of the effects of EZL on the different life cycle stages of Eimeria tenella. EZL mainly acted on the schizogony stage, with peak activity during the second-generation merozoite stage. We also studied the possible target of EZL by identifying the majorly differentially expressed gene affected by EZL in second-generation merozoites using real-time polymerase chain reaction, and screening for surface antigen proteins (SAGs). The relative expression levels of SAGs were compared by Western blot analysis showing that expression levels of surface antigen family member (SAGfm) and SAG19 were significantly downregulated by EZL. Immunofluorescence analysis indicated that SAGfm and SAG19 were localized on the surface of second-generation merozoites. In addition, fluorescence signals were significantly stronger in second-generation merozoites of infected non-medicated control (INC) group compared with that of the EZL group. Therefore, it was speculated that SAGs might be a potential target of EZL action. The inhibitory effects of anticoccidial drugs on SAG levels in coccidia thus warrant further research.

Ethanamizuril (EZL) is a novel anti-coccidiosis triazine compound synthesized in our laboratory. The marker residue of EZL in chicken tissues was EZL and its metabolite deacetylethanamizuril (deacetylEZL). In order to quantitatively analyze the EZL and deacetylEZL in chicken tissues, a rapid, simple, and specific ultra-high-performance liquid chromatography–mass spectrometry (UPLC-MS/MS) method was developed. Validation was performed according to the European Commission Decision 2002/657/EC guidelines. The detection limit was 5 μg kg−1, 10 μg kg−1, and 5 μg kg−1 in muscle, liver, and skin+fat for EZL. And the detection limit of deacetylEZL was 2 μg kg−1, 10 μg kg−1, 2 μg kg−1, and 10 μg kg−1 in muscle, liver, skin+fat, and kidney, respectively. The mean recoveries from fortified samples ranged from 80.15 to 99.50%, with inter assay coefficients of variation ranging between 5.03 and 15.84%. Based on this method, the depletion profile of EZL was studied in 60 healthy chickens after administration of 10 mg L−1 via drinking water for 3 days. Six medicated and one control chicken were sacrificed at 4, 8, 12, 24, 48, 60, 72, 96, and 120 h after the cessation of treatment. The highest residue concentrations of marker residue were attained at the first day, and those of all samples were below the MRL at 68.74 h post-treatment. The concentration of marker residue in kidney tissue was significantly higher than those in liver, skin+fat, and muscle tissues. The statistically estimated withdrawal period of EZL with oral administration was 4 days.

Polysaccharide was derived from Pueraria lobata (PPL) which was considered as one of the traditional Chinese medicinal and edible herbs. In the present study, PPL was administered in equal doses (12.5 mg/kg) to both normal mice and antibiotic-associated diarrhea (AAD) mice for two weeks, and was evaluated in terms of body weight, organ indices, gut structure, gut microbiota and short chain fatty acids. The results showed that normal mice treated with PPL not only reduced the isovaleric acid concentration (P < 0.05), but also significantly increased the abundance of beneficial bacteria, involving Oscillospira and Anaerotruncus (P < 0.05). In addition, PPL could relieve colonic pathological changes and gut microbiota dysbiosis caused by AAD. It indicated that PPL was a potential functional food ingredient by modulating gut microbiota.