Although many mutations contributing to antibiotic resistance have been identified, the relationship between the mutations and the related phenotypic changes responsible for the resistance has yet to ...be fully elucidated. To better characterize phenotype-genotype mapping for drug resistance, here we analyse phenotypic and genotypic changes of antibiotic-resistant Escherichia coli strains obtained by laboratory evolution. We demonstrate that the resistances can be quantitatively predicted by the expression changes of a small number of genes. Several candidate mutations contributing to the resistances are identified, while phenotype-genotype mapping is suggested to be complex and includes various mutations that cause similar phenotypic changes. The integration of transcriptome and genome data enables us to extract essential phenotypic changes for drug resistances.
Behavioral neuroendocrinology has benefited tremendously from the use of a wide range of model organisms that are ideally suited for particular questions. However, in recent years the ability to ...manipulate the genomes of laboratory strains of mice has led to rapid advances in our understanding of the role of specific genes, circuits and neural populations in regulating behavior. While genome manipulation in mice has been a boon for behavioral neuroscience, the intensive focus on the mouse restricts the diversity in behavioral questions that can be investigated using state-of-the-art techniques. The CRISPR/Cas9 system has great potential for efficiently generating mutants in non-traditional animal models and consequently to reinvigorate comparative behavioral neuroendocrinology. Here we describe the efficient generation of oxytocin receptor (Oxtr) mutant prairie voles (Microtus ochrogaster) using the CRISPR/Cas9 system, and describe initial behavioral phenotyping focusing on behaviors relevant to autism. Oxtr mutant male voles show no disruption in pup ultrasonic vocalization, anxiety as measured by the open field test, alloparental behavior, or sociability in the three chamber test. Mutants did however show a modest elevation in repetitive behavior in the marble burying test, and an impairment in preference for social novelty. The ability to efficiently generate targeted mutations in the prairie vole genome will greatly expand the utility of this model organism for discovering the genetic and circuit mechanisms underlying complex social behaviors, and serves as a proof of principle for expanding this approach to other non-traditional model organisms.
•We successfully generated Oxtr gene modified prairie voles using CRISPR/Cas9.•OxtrΔ60/Δ60 prairie voles showed a complete absence of OXTR binding in brain.•Male OxtrΔ60/Δ60 prairie voles showed abnormal repetitive behavior in the marble burying test.•Male OxtrΔ60/Δ60 prairie voles showed impaired social novelty but normal sociability.•Genome editing will enhance the utility of prairie voles for studying mechanisms of behavior.
The emergence and spread of antibiotic resistance in bacteria is becoming a global public health problem. Combination therapy, i.e., the simultaneous use of multiple antibiotics, is used for ...long-term treatment to suppress the emergence of resistant strains. However, the effect of the combinatorial use of multiple drugs on the development of resistance remains elusive, especially in a quantitative assessment.
To understand the evolutionary dynamics under combination therapy, we performed laboratory evolution of Escherichia coli under simultaneous addition of two-drug combinations. We demonstrated that simultaneous addition of a certain combinations of two drugs with collateral sensitivity to each other could suppress the acquisition of resistance to both drugs. Furthermore, we found that the combinatorial use of enoxacin, a DNA replication inhibitor, with Chloramphenicol can accelerate acquisition of resistance to Chloramphenicol. Genome resequencing analyses of the evolved strains suggested that the acceleration of resistance acquisition was caused by an increase of mutation frequency when enoxacin was added.
Integration of laboratory evolution and whole-genome sequencing enabled us to characterize the development of resistance in bacteria under combination therapy. These results provide a basis for rational selection of antibiotic combinations that suppress resistance development effectively.
The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed ...differentiation of human iPSCs into airway basal cells (“iBCs”), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.
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•Directed differentiation of human iPSCs generates airway basal cells (“iBCs”)•iBCs self-renew and display multipotent differentiation in vitro and in vivo•By single-cell RNA-seq, iBCs are highly similar to adult primary airway basal cells•iBCs enable modeling of acquired and genetic airway diseases
Hawkins and colleagues report a directed differentiation protocol enabling the derivation of airway basal cells (“iBCs”) from human iPSCs. iBCs recapitulate hallmark stem cell properties of primary basal cells, including self-renewal and multi-lineage differentiation, thus enabling modeling of airway diseases in vitro and repopulation of tracheal xenografts in vivo.
•We investigated how MS affects the mPFC linked to social behavior using rodents.•MS decreased the number of inhibitory neurons and synapses in the mPFC.•MS impaired the social behavior deeply ...related to the mPFC.•MS attenuated cell activity in the mPFC during social recognition task.•Social deficits by MS is likely to be caused by E/I imbalance in the mPFC.
Aversive environmental conditions during early life are known to cause long-lasting social deficits, similar to those observed in patients with neurodevelopmental disorders. However, the mechanism of how early life stress can cause social deficits is not well understood. To clarify how being in an aversive environment during development affects sociability, we conducted various analyses focusing on the excitatory and inhibitory (E/I) balance in the medial prefrontal cortex (mPFC) and how it is related to social deficits, with young adult male rats that had been exposed to maternal separation (MS). In our MS procedure, part of the pups were separated from each dam for 3 h, twice a day, during postnatal days 2–20, and then were used for each analysis at 9 weeks old. We identified that MS mainly reduced pre- and post-synaptic protein expression of inhibitory neurons in the mPFC, and that decreased the number of GAD67-positive interneurons and inhibitory synapses in the mPFC. Furthermore, MS impaired social behavior related to social recognition, which is closely linked to the mPFC, in the three-chamber sociability and social novelty test (3-CST). With relation to this social deficit, immunohistological analysis revealed that c-fos-positive cells in the mPFC of rats exposed to MS decreased during the 3-CST. Considering that inhibitory neurons in the mPFC play a role in synchronizing neural activation for information processing, our findings demonstrate that MS-induced E/I imbalance associated with cell activity in the mPFC leads to deficits in social recognition.
The corneal endothelium maintains corneal transparency; consequently, its dysfunction causes severe vision loss. Tissue engineering-based therapy, as an alternative to conventional donor corneal ...transplantation, is anticipated to provide a less invasive and more effective therapeutic modality. We conducted a preclinical study for cell-based therapy in a primate model and demonstrated regeneration of the corneal endothelium following injection of cultured monkey corneal endothelial cells (MCECs) or human CECs (HCECs), in combination with a Rho kinase (ROCK) inhibitor, Y-27632, into the anterior chamber. We also evaluated the safety and efficacy of Good Manufacturing Practice (GMP)-grade HCECs, similar to those planned for use as transplant material for human patients in a clinical trial, and we showed that the corneal endothelium was regenerated without adverse effect. We also showed that CEC engraftment is impaired by limited substrate adhesion, which is due to actomyosin contraction induced by dissociation-induced activation of ROCK/MLC signaling. Inclusion of a ROCK inhibitor improves efficiency of engraftment of CECs and enables cell-based therapy for treating corneal endothelial dysfunction as a clinically relevant therapy.
Chromosome damage combined with defective recombinase activity renders cells inviable, owing to deficient double-strand break repair. Despite this, recA polA cells grow well under either DNA damage ...response (SOS) conditions or catalase medium supplementation. Catalase treatments reduce intracellular reactive oxygen species (ROS) levels, suggesting that recA polA cells are susceptible to not only chronic chromosome damage but also ROS. In this study, we used a reducing agent, vitamin C, to confirm whether cell growth could be improved. Vitamin C reduced ROS levels and rescued colony formation in recAts polA cells under restrictive temperatures in the presence of hslO, the gene encoding a redox molecular chaperone. Subsequently, we investigated the role of hslO in the cell growth failure of recAts polA cells. The effects of vitamin C were observed in hslO+ cells; simultaneously, cells converged along several ploidies likely through a completion of replication, with the addition of vitamin C at restrictive temperatures. These results suggest that HslO could manage oxidative stress to an acceptable level, allowing for cell division as well as rescuing cell growth. Overall, ROS may regulate several processes, from damage response to cell division. Our results provide a basis for understanding the unsolved regulatory interplay of cellular processes.
The human Major Histocompatibility Complex (MHC) or Human Leukocyte Antigen (HLA) super-locus is a highly polymorphic genomic region that encodes more than 140 coding genes including the ...transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. In recent years, MHC genotyping and haplotyping using Sanger sequencing and next-generation sequencing (NGS) methods have produced many hundreds of genomic sequences of the HLA super-locus for comparative studies of the genetic architecture and diversity between the same and different haplotypes. In this special issue on 'The Current Landscape of HLA Genomics and Genetics', we provide a short review of some of the recent analytical developments used to investigate the SNP polymorphisms, structural variants (indels), transcription and haplotypes of the HLA super-locus. This review highlights the importance of using reference cell-lines, population studies, and NGS methods to improve and update our understanding of the mechanisms, architectural structures and combinations of human MHC genomic alleles (SNPs and indels) that better define and characterise haplotypes and their association with various phenotypes and diseases.
Three novel HLA‐A*02:06:01:04, ‐DRB1*12:02:01:02, ‐DQB1*03:01:01:07 and five extended HLA‐B*51:01:05, ‐DRB1*12:02:01:01, ‐DRB1*14:01:01, ‐DRB1*14:04:01:01, ‐DRB1*15:04 alleles.
Understanding ethanol tolerance in microorganisms is important for the improvement of bioethanol production. Hence, we performed parallel-evolution experiments using Escherichia coli cells under ...ethanol stress to determine the phenotypic changes necessary for ethanol tolerance.
After cultivation of 1,000 generations under 5% ethanol stress, we obtained 6 ethanol-tolerant strains that showed an approximately 2-fold increase in their specific growth rate in comparison with their ancestor. Expression analysis using microarrays revealed that common expression changes occurred during the adaptive evolution to the ethanol stress environment. Biosynthetic pathways of amino acids, including tryptophan, histidine, and branched-chain amino acids, were commonly up-regulated in the tolerant strains, suggesting that activating these pathways is involved in the development of ethanol tolerance. In support of this hypothesis, supplementation of isoleucine, tryptophan, and histidine to the culture medium increased the specific growth rate under ethanol stress. Furthermore, genes related to iron ion metabolism were commonly up-regulated in the tolerant strains, which suggests the change in intracellular redox state during adaptive evolution.
The common phenotypic changes in the ethanol-tolerant strains we identified could provide a fundamental basis for designing ethanol-tolerant strains for industrial purposes.
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