Damage dynamics and the role of chance in the timing of E. coli cell death.

Genetically identical cells in the same stressful condition die at different times. The origin of this stochasticity is unclear; it may arise from different initial conditions that affect the time of demise, or from a stochastic damage accumulation mechanism that erases the initial conditions and instead amplifies noise to generate different lifespans. To address this requires measuring damage dynamics in individual cells over the lifespan, but this has rarely been achieved. Here, we used a microfluidic device to measure membrane damage in 635 carbon-starved Escherichia coli cells at high temporal resolution. We find that initial conditions of damage, size or cell-cycle phase do not explain most of the lifespan variation. Instead, the data points to a stochastic mechanism in which noise is amplified by a rising production of damage that saturates its own removal. Surprisingly, the relative variation in damage drops with age: cells become more similar to each other in terms of relative damage, indicating increasing determinism with age. Thus, chance erases initial conditions and then gives way to increasingly deterministic dynamics that dominate the lifespan distribution.

Spatial engineering of E. coli with addressable phase-separated RNAs.

Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.

Low-cost anti-mycobacterial drug discovery using engineered E. coli.

Whole-cell screening for Mycobacterium tuberculosis (Mtb) inhibitors is complicated by the pathogen's slow growth and biocontainment requirements. Here we present a synthetic biology framework for assaying Mtb drug targets in engineered E. coli. We construct Target Essential Surrogate E. coli (TESEC) in which an essential metabolic enzyme is deleted and replaced with an Mtb-derived functional analog, linking bacterial growth to the activity of the target enzyme. High throughput screening of a TESEC model for Mtb alanine racemase (Alr) revealed benazepril as a targeted inhibitor, a result validated in whole-cell Mtb. In vitro biochemical assays indicated a noncompetitive mechanism unlike that of clinical Alr inhibitors. We establish the scalability of TESEC for drug discovery by characterizing TESEC strains for four additional targets.

Observing Nutrient Gradients, Gene Expression and Growth Variation Using the "Yeast Machine" Microfluidic Device.

The natural environment of microbial cells like bacteria and yeast is often a complex community in which growth and internal organization reflect morphogenetic processes and interactions that are dependent on spatial position and time. While most of research is performed in simple homogeneous environments (e.g., bulk liquid cultures), which cannot capture full spatiotemporal community dynamics, studying biofilms or colonies is complex and usually does not give access to the spatiotemporal dynamics at single cell level. Here, we detail a protocol for generation of a microfluidic device, the "yeast machine", with arrays of long monolayers of yeast colonies to advance the global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined and customizable spatial dimensions. With Saccharomyces cerevisiae as a model yeast system we used the "yeast machine" to demonstrate the emergence of glucose gradients by following expression of fluorescently labelled hexose transporters. We further quantified the expression spatial patterns with intra-colony growth rates and expression of other genes regulated by glucose availability. In addition to this, we showed that gradients of amino acids also form within a colony, potentially opening similar approaches to study spatiotemporal formation of gradients of many other nutrients and metabolic waste products. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development, and morphogenesis in other same species or more complex multi-species systems at single-cell resolution and timescales relevant to ecology and evolution.

A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies.

Microbial colonies are fascinating structures in which growth and internal organization reflect complex morphogenetic processes. Here, we generated a microfluidics device with arrays of long monolayer yeast colonies to further global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined boundary conditions. We observed the emergence of stable glucose gradients using fluorescently labeled hexose transporters and quantified the spatial correlations with intra-colony growth rates and expression of other genes regulated by glucose availability. These landscapes depended on the external glucose concentration as well as secondary gradients, for example amino acid availability. This work demonstrates the regulatory genetic networks governing cellular physiological adaptation are the key to internal structuration of cellular assemblies. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development and morphogenesis in more complex systems.

Two stochastic processes shape diverse senescence patterns in a single-cell organism.

Despite advances in aging research, a multitude of aging models, and empirical evidence for diverse senescence patterns, understanding of the biological processes that shape senescence is lacking. We show that senescence of an isogenic Escherichia coli bacterial population results from two stochastic processes. The first process is a random deterioration process within the cell, such as generated by random accumulation of damage. This primary process leads to an exponential increase in mortality early in life followed by a late age mortality plateau. The second process relates to the stochastic asymmetric transmission at cell fission of an unknown factor that influences mortality. This secondary process explains the difference between the classical mortality plateaus detected for young mothers' offspring and the near nonsenescence of old mothers' offspring as well as the lack of a mother-offspring correlation in age at death. We observed that lifespan is predominantly determined by underlying stochastic stage dynamics. Surprisingly, our findings support models developed for metazoans that base their arguments on stage-specific actions of alleles to understand the evolution of senescence. We call for exploration of similar stochastic influences that shape aging patterns beyond simple organisms.

Mutation dynamics and fitness effects followed in single cells.

Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli, we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions.

Discovery and Function of a General Core Hormetic Stress Response in E. coli Induced by Sublethal Concentrations of Antibiotics.

A better understanding of the impact of antibiotics on bacteria is required to increase the efficiency of antibiotic treatments and to slow the emergence of resistance. Using Escherichia coli, we examined how bacteria exposed to sublethal concentrations of ampicillin adjust gene expression patterns and metabolism to simultaneously deal with the antibiotic-induced damage and maintain rapid growth. We found that the treated cells increased energy production, as well as translation and macromolecular repair and protection. These responses are adaptive, because they confer increased survival not only to lethal ampicillin treatment but also to non-antibiotic lethal stresses. This robustness is modulated by nutrient availability. Because different antibiotics and other stressors induce the same set of responses, we propose that it constitutes a general core hormetic stress response. It is plausible that this response plays an important role in the robustness of bacteria exposed to antibiotic treatments and constant environmental fluctuations in natural environments.

A synthetic growth switch based on controlled expression of RNA polymerase.

The ability to control growth is essential for fundamental studies of bacterial physiology and biotechnological applications. We have engineered an Escherichia coli strain in which the transcription of a key component of the gene expression machinery, RNA polymerase, is under the control of an inducible promoter. By changing the inducer concentration in the medium, we can adjust the RNA polymerase concentration and thereby switch bacterial growth between zero and the maximal growth rate supported by the medium. We show that our synthetic growth switch functions in a medium-independent and reversible way, and we provide evidence that the switching phenotype arises from the ultrasensitive response of the growth rate to the concentration of RNA polymerase. We present an application of the growth switch in which both the wild-type E. coli strain and our modified strain are endowed with the capacity to produce glycerol when growing on glucose. Cells in which growth has been switched off continue to be metabolically active and harness the energy gain to produce glycerol at a twofold higher yield than in cells with natural control of RNA polymerase expression. Remarkably, without any further optimization, the improved yield is close to the theoretical maximum computed from a flux balance model of E. coli metabolism. The proposed synthetic growth switch is a promising tool for gaining a better understanding of bacterial physiology and for applications in synthetic biology and biotechnology.

[The design and application of anterior cervical pedicle screw-plate system in lower cervical spine].

OBJECTIVE: To explore the applied feasibility of the anterior cervical pedicle screw-plate system in lower cervical spine,in order to provide basic data for clinical application. METHODS: Total thirty-two units (functional spinal unit, FSU) were got randomly from 16 cervical speciments, 8 units in each group of C3,4, C4,5, C5,6 and C6,7. The anterior cervical pedicle screw-plate system was implanted to reconstruct the stability of FSU after discectomy and bone graft. The adaptability was measured between the screw-plate system and vertebral body. X-ray and CT were used to evaluate the accuracy of anterior cervical pedicle screws. The subject will be dissected to identify the situation of involvement if screw perforating the pedicle. RESULTS: Sixty-four anterior pedicle screws were inserted smoothly in the 32 units. The screw and the plate were harmonious locked in the system. The position and length of all screws were satisfactory through X-ray views. However,6 screws perforated the transpedicular (degree 1) according to CT axial views,2 internally cortex and 4 laterally cortex. None perforation was degree 2 or more. None cervical sac compression and nerve root injury was observed in two internal perforation cadavers. One vertebral vein involvement was found in the four lateral perforation screws. The vertebral artery was not pinched though one screw near to the artery. CONCLUSION: The anterior cervical pedicle screw-plate system is adapted to reconstruct in lower cervical spine and it deserved to be used for clinical application.

Direct assessment in bacteria of prionoid propagation and phenotype selection by Hsp70 chaperone.

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria. Using time-lapse microscopy and a microfluidic set-up, we have assessed in Escherichia coli the vertical transmission of the amyloidosis caused by the synthetic bacterial model prionoid RepA-WH1 at single cell resolution within their lineage context. We identify in vivo the coexistence of two strain-like types of amyloid aggregates within a genetically identical population and a controlled homogeneous environment. The amyloids are either toxic globular particles or single comet-shaped aggregates that split during cytokinesis and exhibit milder toxicity. Both segregate and propagate in sublineages, yet show interconversion. ClpB (Hsp104) chaperone, key for spreading of yeast prions, has no effect on the dynamics of the two RepA-WH1 aggregates. However, the propagation of the comet-like species is DnaK (Hsp70)-dependent. The bacterial RepA-WH1 prionoid thus provides key qualitative and quantitative clues on the biology of intracellular amyloid proteinopathies.

[Analysis of epidural hematoma formative reason and its preventive measure after anterior cervical operation].

OBJECTIVE: To explore the risk factors,preventive measure of epidural hematoma after anterior cervical operation. METHODS: From June 2005 and December 2012, 1,452 patients underwent anterior cervical operation in our hospital. Epidural hematoma occurred in 5 cases after operation and the incidence rate was 0.34%. There were 4 males and 1 female with an average age of 46.4 years (ranged, 33 to 55); 3 cases with cervical myelopathy, 1 case with cervical myelopathy and C5 vertebral angeioma, 1 case with ossification of cervical posterior longitudinal ligament. The occurred time,main clinical situation,duration of symptoms,operative management of epidural hematoma were analyzed. RESULTS: Five patients with epidural hematoma occurred within 24 h; the average interval between onset of symptoms and surgery was 4 h (ranged, 2 to 7). Operative treatment was accomplished in 5 cases by exploration and hematoma evacuation. There was significant improvement in all patients after reoperation. Epidural hematoma occurred again in one patient at 5 h after hematoma evacuation, and reoperation were performed to treat it. All patients were followed up from 6 to18 months with an average of 13.8 months. No recurrence was found. CONCLUSION: Intensive care in 24 h postoperatively is important because of epidural hematoma often occurs in this period,especialy in the period of 6-8 h postoperativey. Clinical findings and MRI can early diagnose epidural hematoma and help treatment. Once it is identified and surgical evacuation would be performed on time.

[Case-control study on injured vertebra pedicle instrumentation and injured vertebra bone grafting for the treatment of thoracolumbar fractures].

OBJECTIVE: To compare the clinical effects of injured vertebra pedicle instrumentation and injured vertebra bone grafting in treating thoracolumbar fractures. METHODS: A retrospective study was performed on 48 patients with single thoracolumbar fractures (type A3) from August 2008 to August 2010. Twenty-four patients were treated with injured vertebra pedicle instrumentation (group A) and 24 were treated with injured vertebra bone grafting (group B). There were 14 males and 10 females with an average age of (44.0 +/- 7.4) years old (34 to 56) in group A and there were 13 males and 11 females with an average age of (42.5 +/- 7.1) years(ranged, 31 to 54) in group B. Operation time, volume of blood loss, complications and the relative parameter of imageology were compared between two groups. RESULTS: There was no significant difference in gender,age, position of injury, volume of blood loss between two groups. Operation time of group A was shorter than that of group B. Cobb angle and injured vertebral height obviously improved at the immediately postoperatively between two groups; there was no significant difference in group A between the immediately and three months postoperatively, but there was significant difference in group B; there was no significant difference between three months and one year postoperatively in two groups. The failure rate of group B was significantly higher than that of group A. CONCLUSION: Pedicle screw fixation in the injured vertebrae has advantage of short operation time,can obtain satisfactory effects and is better than injured vertebra bone grafting in maintaining the reduction in treating single thoracolumbar fractures.