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).     

Seroepidemiological studies of El Tor cholera in Bangladesh: association of serum antibody levels with protection

In rural Bangladesh, family contacts of patients with cholera were studied prospectively to examine whether protection against colonization and disease due to Vibrio cholerae O1 was associated with circulating antibodies to V. cholerae. Family contacts (1,071) of 370 patients with cholera were visited daily for 10 days, cultured for V. cholerae, and queried about diarrhea. Sera collected on days 1 and 21 were assayed for vibriocidal antibodies, IgG and IgA antibodies to cholera toxin, and IgG antibodies to lipopolysaccharide (LPS). Vibriocidal titers of greater than or equal to 20 present in 50% of contacts by 20 years of age were associated with protection against both colonization and disease. An elevated level of IgG antitoxin was not associated with protection against colonization or disease but was the most sensitive indicator of recent symptomatic cholera and of immune response to the oral immunogen B subunit. IgG antibody to LPS and IgA antitoxin were of little value in predicting colonization or disease.     

Effect of misoprostol (PGE1) on glucose metabolism in type-2-diabetic and control subjects

In vitro and in vivo studies have demonstrated that prostaglandins of the E series enhance muscle glucose uptake. We examined the effect of acute misoprostol (PGE1) administration on whole body insulin-mediated glucose disposal, as well as the major intracellular pathways of glucose metabolism in type 2 diabetic (n = 10) and non-diabetic (n = 4) subjects. Each subject received two 240-min euglycaemic insulin (40 mU/m²/min) clamp studies with tritiated glucose and indirect calorimetry. During one of the insulin clamp studies, 200 µg of misoprostol was ingested at 90 and 150 min after the start of the insulin infusion. Insulin-mediated total body glucose disposal, glycolysis, glycogenesis and glucose oxidation were similar during the insulin clamp studies performed without and with misoprostol in both the diabetic and non-diabetic groups. These results demonstrate that the acute administration of misoprostol does not enhance insulin-mediated glucose disposal in either type-2-diabetic or non-diabetic subjects.     

Treatment of end-stage diabetic nephropathy: over a decade of experience at one institution

Results of treatment of end-stage renal disease in 139 patients with diabetes mellitus revealed survival of 76% at 1 year and 48% at 5 years. These results compare favorably with other reports from Europe and the United States, probably because of the greater number of patients receiving renal transplants, and possibly because of the use of continuous ambulatory peritoneal dialysis as a recent treatment modality. Patients not receiving transplants were much older (mean age, 47.8 years) than those receiving transplants. Of those not given transplants, survival was best on CAPD. Comparison of those surviving at least 3 years was made with those expiring in the first year. Long-term survivors were younger, had diabetes for a shorter period, but had higher mean blood pressures and serum creatinine values than short-term survivors. Short-term survivors also had over a 50% incidence of prior myocardial infarction or cardiorespiratory arrest, while no long-term survivors had such a history. Long-term survivors were also more likely to have received a transplant, and short-term survivors were more likely to have received intermittent peritoneal dialysis or hemodialysis. A transplant from a living related donor is the treatment of choice for diabetics under age 40 and perhaps for older patients as well. The choice among CAPD, hemodialysis and cadaver transplant requires consideration of many factors.     

Glucagon-like peptide-1 receptor agonists as neuroprotective agents for ischemic stroke: a systematic scoping review

Stroke mortality and morbidity is expected to rise. Despite considerable recent advances within acute ischemic stroke treatment, scope remains for development of widely applicable neuroprotective agents. Glucagon-like peptide-1 receptor agonists (GLP-1RAs), originally licensed for the management of Type 2 Diabetes Mellitus, have demonstrated pre-clinical neuroprotective efficacy in a range of neurodegenerative conditions. This systematic scoping review reports the pre-clinical basis of GLP-1RAs as neuroprotective agents in acute ischemic stroke and their translation into clinical trials. We included 35 pre-clinical studies, 11 retrospective database studies, 7 cardiovascular outcome trials and 4 prospective clinical studies. Pre-clinical neuroprotection was demonstrated in normoglycemic models when administration was delayed by up to 24 h following stroke induction. Outcomes included reduced infarct volume, apoptosis, oxidative stress and inflammation alongside increased neurogenesis, angiogenesis and cerebral blood flow. Improved neurological function and a trend towards increased survival were also reported. Cardiovascular outcomes trials reported a significant reduction in stroke incidence with semaglutide and dulaglutide. Retrospective database studies show a trend towards neuroprotection. Prospective interventional clinical trials are on-going, but initial indicators of safety and tolerability are favourable. Ultimately, we propose that repurposing GLP-1RAs is potentially advantageous but appropriately designed trials are needed to determine clinical efficacy and cost-effectiveness.     

Calcium sensitivity during staircase with sequential incompletely fused contractions

Activity dependent potentiation is thought to result from phosphorylation of the regulatory light chains of myosin, increasing Ca²⁺ sensitivity. Yet, Ca²⁺ sensitivity decreases early in a period of intermittent contractions. The purpose of this study was to investigate the early change in Ca²⁺ sensitivity during intermittent submaximal tetanic contractions. Flexor digitorum brevis muscle fibres were dissected from mice after cervical disarticulation. Fibres were superfused with Tyrode solution at 32 °C. Length was set to yield maximal tetanic force. Indo-1 was microinjected into fibres and allowed to dissipate for 30 min. Fluorescence was measured at 405 and 495 nm wavelength and the ratio was used to estimate [Ca²⁺]. A control force-Ca²⁺ relationship was determined with stimulation over a range of frequencies, yielding constants for slope, max force, and half-maximal [Ca²⁺] (pCa^2?+₅₀). Data were collected for sequential contractions at 40 Hz at 2 s intervals. Active force decreased over the first 1–4 contractions then increased. A force-pCa²⁺ curve was fit to each contraction, using the control values for the Hill slope and max force by adjusting pCa²⁺₅₀ until the curve passed through the target contraction. Data are presented for three contractions for each fibre: first, maximum shift to the right, and last contraction. There was a significant shift to the right for pCa²⁺₅₀ (decreased Ca²⁺ sensitivity), usually early in the series of intermittent contractions, then pCa^2?+₅₀ shifted to the left, but remained significantly different from the control value. Although potentiation is associated with increased Ca²⁺ sensitivity, this increase begins only after Ca²⁺ sensitivity has decreased and, in most cases, Ca²⁺ sensitivity does not increase above the control level.

     

Persistent reversed end diastolic flow in the fetal middle cerebral artery: an ominous finding

Fetal persistent middle cerebral artery reversed end diastolic flow is a rare and ominous finding. Previous cases have been associated with intracranial hemorrhage, growth restriction, anaemia, and hepatic anomaly. Intrauterine demise or early neonatal death is a common outcome. We report the case of persistent middle cerebral artery reversed end diastolic flow in a well-grown fetus at 32 weeks’ gestation resulting from acute, severe anaemia due to a large feto-maternal hemorrhage. An emergency cesarean section was performed and the neonate required advanced resuscitation and immediate blood transfusion. Postnatal magnetic resonance imaging confirmed a hemorrhagic parietal infarct and bilateral ischaemic changes in the basal ganglia. This provides further evidence that persistent middle cerebral artery reversed end diastolic flow in any fetus is an ominous finding warranting urgent diagnostic evaluation and/or delivery.     

Autologous bone marrow transplantation for acute myeloid leukemia using busulfan plus etoposide as a preparative regimen

We have studied the use of a new preparative regimen for the treatment of patients in remission of acute myeloid leukemia (AML) with autologous bone marrow transplantation. Chemotherapy consisted of busulfan 1 mg/kg every 6 hours for 4 days (total dose, 16 mg/kg) on days -7 through -4 followed by an intravenous infusion over 6 to 10 hours of etoposide 60 mg/kg on day -3. Autologous bone marrow, treated in vitro with 100 micrograms/mL of 4-hydroperoxycyclophosphamide, was infused on day 0. We have treated 58 patients up to the age of 60 years, 32 in first remission, 21 in second or third remission, and 5 with primary refractory AML unresponsive to high-dose Ara-C, but achieving remission with aggressive salvage regimens. Of the first remission patients, there has been 1 treatment related death and 5 relapses. With median follow-up of 22 months, the actuarial relapse rate is 22% +/- 9% and disease-free survival is 76% +/- 9% at 3 years. Patients with favorable French-American-British (FAB) subtypes (M3 or M4 EO) did especially well, with no relapses seen in 15 patients observed for a median of 30 months. Actuarial relapse rate at 3 years was 48% for first remission patients with less favorable FAB subtypes. Of patients in second or third remission, there were 5 treatment related deaths and 4 relapses. With median follow-up of 22 months, the actuarial relapse rate is 25% +/- 11% and disease-free survival is 56% +/- 11% at 3 years. Four of five primary refractory patients died during treatment and 1 remains in remission with short follow-up. These preliminary data are very encouraging and, if confirmed, support the use of autologous purged bone marrow transplantation using aggressive preparative regimens as one approach to improve the outcome of adults with AML.