Many anesthetics, including the volatile agent halothane, prolong the decay of GABA A receptor-mediated IPSCs at central synapses. This effect is thought to be a major factor in the production of anesthesia. A variety of different kinetic mechanisms have been proposed for several intravenous agents, but for volatile agents the kinetic mechanisms underlying this change remain unknown. To address this question, we used rapid solution exchange techniques to apply GABA to recombinant GABA A receptors (␣ 1 ␤ 2 ␥ 2s ) expressed in HEK 293 cells, in the absence and presence of halothane. To differentiate between different microscopic kinetic steps that may be altered by the anesthetic, we studied a variety of measures, including peak concentration-response characteristics, macroscopic desensitization, recovery from desensitization, maximal current activation rates, and responses to the low-affinity agonist taurine. Experimentally observed alterations were compared with predictions based on a kinetic scheme that incorporated two agonist binding steps, and open and desensitized states. We found that, in addition to slowing deactivation after a brief pulse of GABA, halothane increased agonist sensitivity and slowed recovery from desensitization but did not alter macroscopic desensitization or maximal activation rate and only slightly slowed rapid deactivation after taurine application. This pattern of responses was found to be consistent with a reduction in the microscopic agonist unbinding rate (k off ) but not with changes in channel gating steps, such as the channel opening rate (␤), closing rate (␣), or microscopic desensitization. We conclude that halothane slows IPSC decay by slowing dissociation of agonist from the receptor. Key words: GABA; halothane; taurine; synaptic inhibition; anesthetics; receptor kineticsThe activity of ligand-gated ion channels can be described in kinetic terms by defining transition rates between individual metastable states of the receptor. Drug action can then be viewed as altering the transition rates between these states, under the assumption that drug binding does not introduce new transitions to the kinetic scheme. Using this approach to study pharmacological modulation of the GABA A receptor, it has been proposed that barbiturates alter transition rates between agonist-bound closed states (Macdonald et al., 1989a;Macdonald and Olsen, 1994), neurosteroids decrease the exit rate from the desensitized state of the receptor (Zhu and Vicini, 1997), and benzodiazepines increase the agonist binding rate (Rogers et al., 1994) or decrease agonist unbinding rate and accelerate desensitization (Mellor and Randall, 1997). In addition, it has been proposed that dephosphorylation of the GABA A receptor reduces the agonist unbinding rate, slowing deactivation and prolonging inhibitory currents (Jones and Westbrook, 1997).The volatile anesthetic halothane prolongs GABA A receptormediated IPSCs (Gage and Robertson, 1985;Mody et al., 1991;Jones and Harrison, 1993;Pearce, 1996), as do a great number o...

The precise mapping of how complex patterns of synaptic inputs are integrated into specific patterns of spiking output is an essential step in the characterization of the cellular basis of network dynamics and function. Relative to other principal neurons of the hippocampus, the electrophysiology of CA1 pyramidal cells has been extensively investigated. Yet, the precise input-output relationship is to date unknown even for this neuronal class. CA1 pyramidal neurons receive laminated excitatory inputs from three distinct pathways: recurrent CA1 collaterals on basal dendrites, CA3 Schaffer collaterals, mostly on oblique and proximal apical dendrites, and entorhinal perforant pathway on distal apical dendrites. We implemented detailed computer simulations of pyramidal cell electrophysiology based on three-dimensional anatomical reconstructions and compartmental models of available biophysical properties from the experimental literature. To investigate the effect of synaptic input on axosomatic firing, we stochastically distributed a realistic number of excitatory synapses in each of the three dendritic layers. We then recorded the spiking response to different stimulation patterns. For all dendritic layers, synchronous stimuli resulted in trains of spiking output and a linear relationship between input and output firing frequencies. In contrast, asynchronous stimuli evoked non-bursting spike patterns and the corresponding firing frequency input-output function was logarithmic. The regular/irregular nature of the input synaptic intervals was only reflected in the regularity of output inter-burst intervals in response to synchronous stimulation, and never affected firing frequency. Synaptic stimulations in the basal and proximal apical trees across individual neuronal morphologies yielded remarkably similar input-output relationships. Results were also robust with respect to the detailed distributions of dendritic and synaptic conductances within a plausible range constrained by experimental evidence. In contrast, the input-output relationship in response to distal apical stimuli showed dramatic differences from the other dendritic locations as well as among neurons, and was more sensible to the exact channel densities.

The plasmid-borne mobilized colistin resistance gene (mcr-1) was discovered in 2015. Subsequently, the rapid horizontal transfer of mcr-1 gene to diverse bacterial species poses a serious threat to public health, which urgently needs the introduction of novel antimicrobial strategies. Therefore, the purpose of this study is to sensitize bacteria to colistin and reduce the propagation of mcr-1 gene by curing mcr-1-harboring plasmid in Escherichia coli (E. coli) using the CRISPR-Cas9 system. Methods: Two sgRNAs specific to mcr-1 gene were designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-mcr was transformed into E. coli carrying pUC19mcr-1 or pHNSHP45, separately. The elimination efficiency in strains was evaluated by PCR and quantitative real-time PCR (qPCR). The antimicrobial susceptibility test was performed using the broth microdilution method. Results: In this study, we constructed the high copy number plasmid pUC19-mcr-1 and recombinant plasmid pCas9-m1 or pCas9-m2, which contain 20 nt or 30 nt sgRNA sequences targeted to mcr-1, respectively. PCR and qPCR results showed that mcr-1-harboring plasmids could be efficiently eliminated, and there was no significant correlation between sgRNA lengths and curing efficiency. However, when comparing restructured high copy number plasmid (pUC19-mcr-1) to natural resistance plasmid (pHNSHP45) in eliminating efficiency, we found that the content of plasmid backbone had an influence on efficiency. Furthermore, the conjugation assays verified that the engineered CRISPR-Cas9 system in bacteria or in bacteria genome can protect the recipient from plasmid-borne mcr-1 transfer via conjugation. Additionally, sequence analysis showed that three different types of defects in CRISPR-Cas9 system lead to escape mutants. Conclusion: We presented a method that only one plasmid-mediated CRISPR-Cas9 system can be used to efficiently resensitize E. coli to colistin. Moreover, this system provided a great potentiality to counteract the propagation of mcr-1 among bacterial pathogens.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic microorganism that is increasingly causing public health concern worldwide. The objective of this study was to determine the transmission and occurrence of MRSA in a slaughterhouse environment and evaluate its antimicrobial resistance and genetic characterization. In this study, we conducted a comprehensive epidemiological survey of S. aureus by spa typing and whole-genome sequencing (WGS) of samples obtained from the pork production chain, the environment, and community residents. To clarify the evolutionary relationships of MRSA sequence type (ST) 398 in this study and global isolates, 197 published whole-genome sequences data of MRSA ST398 strains were downloaded from the GenBank database and included in the phylogenetic analysis. A total of 585 porcine samples (snout and carcass swabs), 78 human nasal samples, and 136 environmental samples were collected. The MRSA isolates were detected at higher frequencies in samples from swine (15.0%) than carcasses (10.0%), slaughterhouse workers (8.0%), community residents (0%), and environment samples (5.9%). The spa typing results showed that t571 accounted for a higher proportion than other spa types. Closely related isolates from the samples of swine, slaughterhouse workers, carcasses, carrier vehicle, and surrounding fishpond water indicate that MRSA ST398 strains may spread among swine, humans, and the environment. MRSA ST398-t571 isolates were genetically different from global strains, except for two Korean isolates, which showed genetic closeness with it. In addition, a MRSA ST398 isolate recovered from an infected patient in Europe differed by only 31 SNPs from the airborne dust-associated strain isolated in this study, thereby suggesting potential transmission among different countries. Antimicrobial susceptibility testing results demonstrated that 99.0% (96/97) of MRSA and 95.1% (231/243) of methicillin-sensitive S. aureus (MSSA) showed multidrug-resistant (MDR) phenotypes. According to WGS analysis, the poxtA-carrying segment (IS431mec-optrA-IS1216-fexB-IS431mec) was reported in MRSA ST398 isolates for the first time. The coexistence of cfr and optrA in a plasmid was first detected in MRSA ST398. The potential transmission of MRSA among humans, animals, and the environment is a cause for concern. The emergence and transmission of LA-MRSA ST398 with high levels of resistance profiles highlight the urgent need for LA-MRSA surveillance.