Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

Microperimetry and spectral domain optical coherence tomography in myelinated retinal nerve fibers

AIM: To complete the data of ocular trauma in central China, as a well-known tertiary referral center for ocular trauma, we documented the epidemiological characteristics and visual outcomes of patients hospitalized for ocular trauma in this region. METHODS: A retrospective study of patients hospitalized for ocular trauma in central China from 2006 to 2011 was performed. RESULTS: This study included 5964 eyes of 5799 patients. The average age was 35.5±21.8y with a male-to-female ratio of 2.8:1. The most common age was 45-59y age group. Most patients were farmers and workers (51.9%). The most common injuries were firework related (24.5%), road traffic related (24.2%), and work related (15.0%). Among the most common causative agents were firecrackers (24.5%), followed by metal/knife/scissors (21.4%). Most injuries occurred in January (14.2%), February (27.0%), and August (10.0%). There were 8.5% patients with ocular injuries combined with other injuries. The incidence of open ocular injuries (4585 eyes, 76.9%) was higher than closed ocular injuries (939 eyes, 15.7%). The incidences of chemical and thermal ocular injuries were 1.2% and 0.6%. Ocular trauma score (OTS) predicted final visual acuity at non light perception (NLP), 20/200-20/50 and 20/40 with a sensitivity of 100%, and light perception (LP)/hand motion (HM) and 1/200-19/200 with a specificity of 100%. CONCLUSIONS: This study provides recent epidemiological data of patients hospitalized for ocular trauma in central China. Some factors influencing the visual outcome include time interval between injury and visit to the clinic, wound location, open or closed globe injury, initial visual acuity, and OTS.     

Autoimmune cholangitis in a patient with thymoma

Autoimmune cholangitis is characterized biochemically by chronic cholestasis and histopathologically by chronic non-suppurative destructive cholangitis. It is associated with positive antinuclear antibody test and negative antimitochondrial antibody test results. Recently, we experienced a case of a 35-year-old woman with autoimmune cholangitis associated with thymoma who presented with pruritis, jaundice, chronic fatigue and anterior chest discomfort. Her laboratory examinations revealed marked increases in levels of serum alkaline phosphatase and gamma-glutamyl transpeptidase. In serological tests, antinuclear antibody was found, but antimitochondrial antibody was not. Liver biopsy findings were compatible with chronic non-suppurative destructive cholangitis. On computed tomography (CT) of the chest, a large anterior mediastinal mass was found. The mass was totally resected and the patient was treated with ursodeoxy cholic acid. Thereafter, her clinical symptoms improved and liver functions completely returned to the normal range. We describe here an uncommon association of autoimmune cholangitis with thymoma, which has not been reported previously in the English-written literature.     

Factors associated with intention to be tested for HIV among men who have sex with men in a country with a very low HIV prevalence

This study examined factors associated with the intention to take an HIV test among men who have sex with men (MSM) in South Korea. An internet website-based survey was conducted among users of the only and largest online MSM website between 20 July 2016, and 20 August 2016. A total of 2915 participants completed the survey and answered questions related to sociodemographic information, health behaviors, sexual behaviors, and HIV testing history. Of these, 2587 (88.7%) participants responded as having an intention to take an HIV test. A multivariable logistic regression analysis revealed the following as having reduced the intention to undergo HIV testing: very good subjective health status and no sexual interactions during the last 6 months (Adjusted odds ratios [AOR] 0.45 and 0.54, respectively). In contrast, increased intention to take an HIV test was associated with being 20–29 years old, 30–39 years old, not paying or receiving money for sex, having a history of HIV testing, and taking an HIV test once per 12 months (AOR 2.64, 2.13, 1.54, 1.81, and 2.17, respectively). In conclusion, HIV testing among MSM in this study was associated with age, subjective health status, sex(es) of one’s sexual partner(s) during the last 6 months, sexual risk behaviors, HIV testing history, and undergoing regular HIV testing.     

Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles

In hair follicle development, a placode-derived signal is believed to induce formation of the dermal condensation, an essential component of ectodermal organs. However, the identity of this signal is unknown. Furthermore, although induction and patterning of hair follicles are intimately linked, it is not known whether the mesenchymal condensation is necessary for inducing the initial epithelial pattern. Here, we show that fibroblast growth factor 20 (Fgf20) is expressed in hair placodes and is induced by and functions downstream from epithelial ectodysplasin (Eda)/Edar and Wnt/β-Catenin signaling to initiate formation of the underlying dermal condensation. Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles and subsequent formation of guard, awl, and auchene hairs. Although primary dermal condensations are absent in Fgf20 mutant mice, a regular array of hair placodes is formed, demonstrating that the epithelial patterning process is independent of known histological and molecular markers of underlying mesenchymal patterns during the initial stages of hair follicle development.     

Functional Differentiation of Mouse Visual Cortical Areas Depends upon Early Binocular Experience

The mammalian visual cortex contains multiple retinotopically defined areas that process distinct features of the visual scene. Little is known about what guides the functional differentiation of visual cortical areas during development. Recent studies in mice have revealed that visual input from the two eyes provides spatiotemporally distinct signals to primary visual cortex (V1), such that contralateral eye-dominated V1 neurons respond to higher spatial frequencies than ipsilateral eye-dominated neurons. To test whether binocular visual input drives the differentiation of visual cortical areas, we used two-photon calcium imaging to characterize the effects of juvenile monocular deprivation (MD) on the responses of neurons in V1 and two higher visual areas, LM (lateromedial) and PM (posteromedial). In adult mice of either sex, we find that MD prevents the emergence of distinct spatiotemporal tuning in V1, LM, and PM. We also find that, within each of these areas, MD reorganizes the distinct spatiotemporal tuning properties driven by the two eyes. Moreover, we find a relationship between speed tuning and ocular dominance in all three areas that MD preferentially disrupts in V1, but not in LM or PM. Together, these results reveal that balanced binocular vision during development is essential for driving the functional differentiation of visual cortical areas. The higher visual areas of mouse visual cortex may provide a useful platform for investigating the experience-dependent mechanisms that set up the specialized processing within neocortical areas during postnatal development.SIGNIFICANCE STATEMENT Little is known about the factors guiding the emergence of functionally distinct areas in the brain. Using in vivo Ca²⁺ imaging, we recorded visually evoked activity from cells in V1 and higher visual areas LM (lateromedial) and PM (posteromedial) of mice. Neurons in these areas normally display distinct spatiotemporal tuning properties. We found that depriving one eye of normal input during development prevents the functional differentiation of visual areas. Deprivation did not disrupt the degree of speed tuning, a property thought to emerge in higher visual areas. Thus, some properties of visual cortical neurons are shaped by binocular experience, while others are resistant. Our study uncovers the fundamental role of binocular experience in the formation of distinct areas in visual cortex.