Dose Assessment of Cefquinome by Pharmacokinetic/Pharmacodynamic Modeling in Mouse Model of Staphylococcus aureus Mastitis.

This work aimed to characterize the mammary gland pharmacokinetics of cefquinome after an intramammary administration and integrate pharmacokinetic/pharmacodynamic model. The pharmacokinetic profiles of cefquinome in gland tissue were measured using high performance liquid chromatograph. Therapeutic regimens covered various dosages ranging from 25 to 800 µg/gland and multiple dosing intervals of 8, 12, and 24 h. The in vivo bacterial killing activity elevated when dosage increased or when dosing intervals were shortened. The best antibacterial effect was demonstrated by a mean 1.5 log10CFU/gland visible count reduction. On the other hand, the results showed that the percentage of time duration of drug concentration exceeding the MIC during a dose interval (%T > MIC) was generally 100% because of the influence of drug distribution caused by the blood-milk barrier. Therefore, pharmacokinetic/pharmacodynamic parameter of the ratio of area under the concentration-time curve over 24 h to the MIC (AUC0-24/MIC) was used to describe the efficacy of cefquinome instead of %T > MIC. When the magnitude of AUC0-24/MIC exceeding 16571.55 h⋅mL/g, considerable activity of about 1.5 log10CFU/g gland bacterial count reduction was observed in vivo. Based on the Monte Carlo simulation, the clinical recommended regimen of three infusions of 75 mg per quarter every 12 h can achieve a 76.67% cure rate in clinical treatment of bovine mastitis caused by Staphylococcus aureus infection.

In Vivo Pharmacokinetics/Pharmacodynamics of Cefquinome in an Experimental Mouse Model of Staphylococcus Aureus Mastitis following Intramammary Infusion.

Staphylococcus aureus remains the major cause of morbidity of bovine mastitis worldwide leading to massive economic losses. Cefquinome is a fourth generation cephalosporin, which preserves susceptibility and antibacterial activity against S. aureus. This work aims to study the pharmacokinetic (PK) and pharmacodynamic (PD) modeling following intramammary administration of cefquinome against S. aureus mastitis. The mouse model of S. aureus mastitis was developed for the PK/PD experiments. The plasma PK characteristics after intramammary injection of cefquinome at various single doses of 25, 50, 100, 200, 400 µg per gland (both fourth pairs of glands: L4 and R4) were calculated using one-compartment and first-order absorption model. PD study was investigated based on twenty-one intermittent dosing regimens, of which total daily dose ranged from 25 to 4800 µg per mouse and dosage intervals included 8, 12 or 24 h. The sigmoid Emax model of inhibitory effect was employed for PK/PD modeling. The results of PK/PD integration of cefquinome against S. aureus suggested that the percentage of duration that drug concentration exceeded the minimal inhibitory concentration (%T>MIC) and the ratio of area under time-concentration curve over MIC (AUC/MIC) are important indexes to evaluate the antibacterial activity. The PK/PD parameters of %T>MIC and AUC0-24/MIC were 35.98% and 137.43 h to obtain a 1.8 logCFU/gland reduction of bacterial colony counts in vivo, against S. aureus strains with cefquinome MIC of 0.5µg/ml.

[AF172993 sequence of Plunc in GenBank database is not the complete CDS].

OBJECTIVE: To determine AF172993 sequence is either the complete CDS or a transcript variant. METHODS: RT-PCR was used to amplify the CDS sequence of Plunc, which was subsequently cloned into the pEGFP-N1 eukaryotic expression vector. After bi-directional sequence analysis, the sequence obtained was blasted against the AF172993 sequence, nr database and human genome database. RESULTS: In CDS of the new cloned sequence, the 658 base A in the AF172993 sequence was replaced by C, and the corresponding genetic code was also converted from AAG to CAG, leading to the alteration of the amino acid Gln to Lys. In addition, the base C at the 658 position of the CDS showed perfect match with the base C at 2094188 position in human chromosome 20. CONCLUSION: The base A at the 658 position of AF172993 sequence of Plunc is a mutation site, which alters the coding of the amino acid. AF172993 sequence is actually a transcript variant of Plunc, and the annotation to AF172993 in GenBank database is not correct and need to be revised.

[A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nuclei].

OBJECTIVE: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.

[Effects of different electrofusion parameters on activation and early development of mouse embryos reconstructed by cumulus cell nuclear transfer].

OBJECTIVE: To study the effects of different parameters for electrofusion on the activation and early development of mouse embryos reconstructed by cumulus cell nuclear transfer, and explore optimal parameters for electrofusion. METHODS: A C57BL/6j mouse cumulus cell nucleus 10-12 mm in diameter was inserted into the perivitelline space of an enucleated oocyte. The fusion of donor-recipient pairs was induced with different parameters for electrofusion (with variation in electric field intensity, pulse duration and pulse times). Successful formation of the reconstructed embryos from donor-recipient pairs and the reconstructed embryos developing into the early embryonic stages (2-cell, 4-8-cell and morula stages) were observed and counted. RESULTS: The electric field intensity and pulse duration allowed variation during electrofusion within the range of 1 000-2 000 kV/cm and 40-160 ms, respectively. The donor-recipient pairs fused at very low rate when the parameters were below the allowed ranges, and disintegration or even death might occur when the parameters were above the ranges. Within these allowed ranges, variation of the electrofusion parameters did not produce significant impact on the ratio of the reconstructed embryos in 2-cell, 4-8-cell or morula stages (P<0.05). In addition, we suggested that pulse times be limited to 1-2. CONCLUSION: Optimal parameters for electrofusion are crucial for the fusion of donor-recipient pairs and the activation of the reconstructed embryo.

[Construction of N-LMP1 transgenic mice with the specific regulation region in nasopharynx].

In order to elucidate the role of EBV-LMP1 in the nasopharyngeal carcinogenesis, the expression vector was constructed with subjecting the N-LMP1 gene to double regulation of two specific regulators: EDL-2 and PLUNC-p. The N-LMP1 related transgenic mice model has been constructed successfully by pronucleus microinjection. 58 founder mice were born, 4 of which were founded to be positive by PCR and Southern blot. Immunohistochemistry assay showed that N-LMP1 protein was expressed in the nasopharynx, tongue and forestomach of transgenic mice.

[Production of transgenic mice by in vivo spermatogonia-mediated gene transfer].

The present study examined if injection of DNA into the testis tabular could generate transgenic mice via transfecting spermatogonia. The 0.9 kb Bcl-2 cDNA was fused to the promoter region of mouse whey acid protein gene and the SV40 polyA. A 3.0 kb fragment of WAP-Bcl-2-SV40 was mixed with cationic liposome and one side tabular of 3 mice testis was injected with the fragment, the other side was ligatured. Two out of the 3 males were used to mate with the female 4 days later. Twenty pups were produced and 3 of which were proven to be gene integration positive by PCR detection. Two, 1 male and 1 female, were further confirmed to carry the transgene by Southern blot analysis. The male died by accident during its feeding. The female was demonstrated to express Bcl-2 protein in its mammary glands by Western blot assay. Seven out of 45 F1 mice were proven to be transgenic by PCR. It is concluded that transfecting spermatogonia in vivo can produce transgenic mice.

[Construction of transgenic mice carrying enhanced green fluorescent protein gene by seminiferous tubule microinjection].

OBJECTIVE: To study the feasibility of establishing transgenic mice carrying enhanced green flourescent protein (EGFP) gene by means of seminiferous tubule microinjection. METHODS: The vector expressing enhanced green fluorescent protein under the control of human cytomegalovirus immediate-early promoter (pCMV-EGFP) was selected and mixed with liposome in vitro. Microinjection at different doses of the liposome-entrapped plasmid DNA into the seminiferous tubules of male mice at different ages was performed to establish transgenic mice, which were made to mate with female mice at least 40 d after the microinjection. Genomic DNA was extracted from the offspring of the founder mice for PCR and Southern blotting analysis, and the frozen sections of different tissues from 2 of the founders mice were prepared for fluorescence microscopic observation. RESULTS: Among the 41 mice receiving the microinjection, 32 survived and retained their mating ability and fertility, and among their 382 offspring 133 were positive for EGFP DNA as demonstrated by PCR, 15 of which were confirmed by Southern blotting analysis. The age of the mice or the doses of microinjection they received was not shown to impact the integration of EGFP gene, and fluorescence microscopy failed to detect significant EGFP expression in the tissues of the founder mice (P>0.05) in comparison with normal mice. CONCLUSION: Seminiferous tubule microinjection is simple and practicable to implement gene transfer in mice.