Three-dimensional characterisation of the globe position in the orbit

Graefe's Archive for Clinical and Experimental Ophthalmology - Current methods to analyse the globe position, including Hertel exophthalmometry and computed tomography (CT), are limited to the...     

Response of Escherichia coli growth rate to osmotic shock

It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless “stored” growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan.Cell growth is the result of a complex system of biochemical processes and mechanical forces. For bacterial, plant, and fungal cells, growth requires both the synthesis of cytoplasmic components and the expansion of the cell wall, a stiff polymeric network that encloses these cells. It is well established that plant cells use turgor pressure, the outward normal force exerted by the cytoplasm on the cell wall, to drive mechanical expansion of the cell wall during growth (1, 2). In contrast, our understanding of the physical mechanisms of cell-wall expansion in bacteria is limited. Furthermore, the bacterial cell wall is distinct from its eukaryotic counterparts in both ultrastructure and chemical composition. In particular, the peptidoglycan cell wall of Gram-negative bacteria is extremely thin, comprising perhaps a molecular monolayer (3). This raises the question of whether these organisms require turgor pressure for cell-wall expansion, or whether they use a different strategy than organisms with thicker walls.Turgor pressure is established within cells according to the Morse equation, P = RT(C_in ? C_out), where C_in is the osmolarity of the cytoplasm, C_out is the osmolarity of the extracellular medium, R is the gas constant, and T is the temperature. In the Gram-negative bacterium Escherichia coli, P has been estimated to be 1–3 atm (4, 5). A primary role of the cell wall is to bear this load by balancing it with mechanical stress, thereby preventing cell lysis. In 1924, Walter proposed a theory of bacterial growth based on the premise that mechanical stress, in turn, is responsible for stretching the cell wall during growth (6). In support of this theory, he and others showed that the growth rate of a number of bacterial species, including E. coli, decreases as the osmolarity of their growth medium is increased (6–9) (Fig. 1A). This result would be predicted by the Morse equation if growth rate, e˙, scaled with pressure: e˙∼P∼−Cout. The decrease in growth rate did not depend on the chemical used to modulate the osmolarity, which demonstrated that this effect was not due to specific, toxic reactions. More recently, several theoretical studies have offered plausible molecular mechanisms by which turgor pressure could drive cell-wall expansion. These theories range from ones in which the cell wall is irreversibly stretched by turgor pressure when peptidoglycan cross-links are hydrolyzed (10, 11) to ones in which the rate of peptidoglycan biosynthesis is directly dependent on turgor pressure (12, 13). Two of these theories explicitly predict the scaling between cellular growth rate and pressure (12, 13), providing possible explanations of the classic experiments (6–9).Open in a separate windowFig. 1.E. coli cells maintain their elongation rate after hyperosmotic shock. (A) The population-averaged steady-state elongation rate as a function of the concentration of sorbitol in the growth medium, C_out (red circles). Also shown are the population-averaged elongation rates during phase III of recovery from a 400 mM hyperosmotic shock (blue triangles) and immediately after a 400 mM hypoosmotic shock (green squares). Each data point is averaged over 20–100 cells. Error bars indicate ±1 SE. (Inset) Definition of elongation rate. (B) E. coli stained with fluorescent WGA, imaged with phase and epifluorescence microscopy. (Lower) Schematic defining the length and width of a rod-shaped cell. (C) Longitudinal strain, ε_l, and radial strain, ε_w, as a function of medium osmolarity. Error bars indicate ±1 SE. (Inset) Tracked outlines of the wall of four cells before (red) and after (blue) plasmolysis. (D) (Left) Initial images of a single cell: phase, epifluorescence, and overlay. In the overlay, the epifluorescence kymograph is false-colored green and the inverse of the phase kymograph is false-colored red. (Right) Kymographs of this cell elongating and dividing during a 400 mM hyperosmotic shock. The kymographs consist of a montage of a one-pixel-wide region around the long axis of the cell, indicated by the dashed line. The white arrow indicates the time of the shock. The red arrow indicates the period of plasmolysis during which the cytoplasm is shorter than the cell wall. The black arrow indicates the formation of a septum during cell division. (E) The concentration of sorbitol in the growth medium versus time during a hyperosmotic shock. (F) The length of the cell walls of representative cells during the same shock. The background colors indicate the four phases of response. (G) The population-averaged elongation rate of both the cytoplasm and the cell wall during the same shock (n = 42 cells). The data were smoothed with a moving-average filter with a 2-min window for illustrative purposes. The blue dotted lines indicate the steady-state elongation rates in LB (upper line) and LB+400 mM sorbitol (lower line). The confidence intervals indicate ±1 SE. The cell wall could not be tracked indefinitely owing to dilution of the cell-wall stain. (Lower) Key describing the four phases of response to hyperosmotic shock.However, bacterial cells possess several mechanisms for regulating their cytoplasmic osmolarity in response to changes in their external osmotic environment (14). Specifically, E. coli imports and synthesizes compatible solutes, and imports ions, in response to high external osmolarities (15). For example, the concentration of potassium ions in the cytoplasm scales as the external osmolality (16). Therefore, it is unclear whether raising medium osmolarity actually causes a decrease in turgor pressure over long time scales. The inverse correlation between growth rate and medium osmolarity could result from another osmotic effect such as altered water potential within the cell, which could affect, for example, protein folding (17) or signaling (18). We sought to distinguish between these possibilities by measuring the elastic strain within the cell wall as a function of medium osmolarity and by determining the time scale over which osmotic shock (acute changes in medium osmolarity) modulates growth rate and cell-wall synthesis. If growth rate scales with turgor pressure, then (i) growth rate should also scale with cell-wall elastic strain, and therefore elastic strain should decrease with increasing medium osmolarity, and (ii) growth rate, and perhaps the rate of cell-wall synthesis, should rapidly change upon osmotic shock. However, if medium osmolarity modulates growth rate independently of pressure, these processes may adapt more slowly to changes in osmolarity.We found that elastic strain decreases only moderately with increasing medium osmolarity. By measuring the growth-rate response of E. coli across time scales that spanned four orders of magnitude we concluded that osmotic shock had little effect on growth rate, except in the case of plasmolysis (when P ≤ 0, causing the cytoplasm to separate from the cell wall). The growth rate of E. coli adapted slowly to changes in medium osmolarity, over the course of tens of minutes. Furthermore, when turgor pressure was restored after slight plasmolysis, E. coli cells quickly elongated to the length that they would have attained had they never been plasmolyzed. From this result, we inferred that peptidoglycan synthesis is insensitive to changes in turgor pressure. In support of this hypothesis, we found that the speed of MreB, a protein whose motion is dependent on peptidoglycan synthesis (19–21), is largely unaffected by osmotic shock. Therefore, our results demonstrate that cell-wall biosynthesis is the rate-limiting factor in cell-wall expansion and that turgor pressure is not required for cell-wall biosynthesis, but that pressure does play a simple role whereby it stretches recently assembled, unextended peptidoglycan.     

The efficacy and safety of intravenous hydralazine for the treatment of hypertension in the hospitalized child

Background

Intravenous (IV) hydralazine is frequently used for the treatment of elevated blood pressure (BP) in hospitalized children. Its safety and efficacy have not been examined.

Methods

This is a retrospective chart review of IV hydralazine use in hospitalized children (birth to 17 years) over a 3-year period. Demographic data and data on adverse effects (AE), BP, and heart rate (HR) prior to and after each first dose were collected.

Results

The patient cohort comprised 110 children admitted to the hospital during the study period, of whom 77 received the recommended dose. Mean age of the children was 8.5?±?5.4 years; 33 % were male, and 32.5 % were white. Pre-dose systolic and diastolic BP indexes were 1.3 and 1.2, respectively. The median reduction in systolic and diastolic BP was 8.5 and 11.5 %, respectively. Sixteen (21 %) children achieved a 25 % reduction in systolic or diastolic BP, and BP increased in 30 % of patients; 10 % of children had a BP of <95th percentile for age, sex, and height after one dose. Seven (9 %) children had a documented AE. HR increased by a median of 3.5 %. In the multivariable models examining percentage change in systolic and diastolic BP, male gender was significantly associated with a change in systolic BP.

Conclusions

In hospitalized children, IV hydralazine was well tolerated, BP response was variable, and 21 % of the patients achieved a ≥25 % reduction of systolic or diastolic BP. Further studies are needed to compare the safety and efficacy of IV hydralazine to other short-acting antihypertensive agents.     

Does additional support by nurses enhance the effect of a brief smoking cessation intervention in people with moderate to severe chronic obstructive pulmonary disease? A randomised controlled trial

BACKGROUND: Smoking cessation is the primary disease modifying intervention for chronic obstructive pulmonary disease (COPD). SETTING: A Regional Respiratory Centre (RRC) out-patient department in Northern Ireland. METHODS: A randomised controlled trial (RCT) evaluated the effectiveness of brief advice alone or accompanied by individual nurse support or group support facilitated by nurses. Smoking status was biochemically validated and stage of change, nicotine addiction and dyspnoea were recorded at 2, 3, 6, 9 and 12 months. PARTICIPANTS: Ninety-one cigarette smokers with COPD were enrolled in the study (mean age 61 years, 47 female). RESULTS: After 12 months cessation rates were not significantly different between groups (p=0.7), but all groups had a significant reduction in their nicotine addiction (p=0.03-0.006). No changes in subjects' motivation or dyspnoea were detected over the 12 months. CONCLUSION: Patients with COPD were unable to stop smoking regardless of the type of support they received. Harm reduction may be a more appropriate goal than complete cessation for intractable smokers and nurses must evaluate their role in this arena.     

A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs

Background and purpose

The osteogenic potency of erythropoietin (EPO) has been documented. However, its efficacy in a large-animal model has not yet been investigated; nor has a clinically safe dosage. The purpose of this study was to overcome such limitations of previous studies and thereby pave the way for possible clinical application. Our hypothesis was that EPO increases calvarial bone healing compared to a saline control in the same subject.

Methods

We used a porcine calvarial defect model. In each of 18 pigs, 6 cylindrical defects (diameter: 1 cm; height: 1 cm) were drilled, allowing 3 pairwise comparisons. Treatment consisted of either 900 IU/mL EPO or an equal volume of saline in combination with either autograft, a collagen carrier, or a polycaprolactone (PCL) scaffold. After an observation time of 5 weeks, the primary outcome (bone volume fraction (BV/TV)) was assessed with high-resolution quantitative computed tomography. Secondary outcome measures were histomorphometry and blood samples.

Results

The median BV/TV ratio of the EPO-treated collagen group was 1.06 (CI: 1.02–1.11) relative to the saline-treated collagen group. Histomorphometry showed a similar median effect size, but it did not reach statistical significance. Autograft treatment had excellent healing potential and was able to completely regenerate the bone defect independently of EPO treatment. Bony ingrowth into the PCL scaffold was sparse, both with and without EPO. Neither a substantial systemic effect nor adverse events were observed. The number of blood vessels was similar in EPO-treated defects and saline-treated defects.

Interpretation

Topical administration of EPO on a collagen carrier moderately increased bone healing. The dosing regime was safe, and could have possible application in the clinical setting. However, in order to increase the clinical relevance, a more potent but still clinically safe dose should be investigated.Erythropoietin (EPO) is a hematopoietic growth factor that stimulates the formation of red blood cells. In recent years, the non-hematopoietic effects of EPO have been investigated. Of interest for skeletal tissue engineering, the pleiotropic capabilities of EPO include osteogenic and angiogenic potencies (Rölfing et al. 2012). Subcutaneous injections of 250 IU/kg EPO were found to enhance bone formation 6 weeks after operation in a spinal fusion model in rabbits (Rölfing et al. 2012). The validity of the methodology of this study was confirmed in a recently published meta-analysis (Riordan et al. 2013, Rölfing and Bünger 2013). Other independent research groups have reported increased bone formation in mice and rats after daily treatment with 200–6,000 IU/kg EPO (Bozlar et al. 2006, Holstein et al. 2007, 2011, Shiozawa et al. 2010, Garcia et al. 2011, Kim et al. 2012). Furthermore, vascularization of 3-dimensional scaffolds for bone tissue regeneration remains a challenge. The described pleiotropic functions of EPO may overcome this limitation of skeletal tissue engineering in the future. EPO could possibly facilitate angiogenesis directed into the core of the scaffold, thereby facilitating bony ingrowth. Moreover, EPO promotes a direct and indirect osteogenic stimulation of mesenchymal stromal cells (Shiozawa et al. 2010, Rölfing et al. 2013).Translation of these promising in vitro and in vivo data into clinical trials requires a physiological dosage of EPO in order to avoid its known complications, such as thromboembolism (Ehrenreich et al. 2009, Shiozawa et al. 2010, Kim et al. 2012, Rölfing et al. 2012). Notably, we observed an extremely high hematocrit level after 250 IU/kg EPO for 20 days in a rabbit model (Rölfing et al. 2012). In other in vivo studies, repetitive EPO injections ranging from 500 to 6,000 IU/kg were administered. These treatment regimes have a systemic effect, and thus hold the risk of adverse events. Testing of the efficacy of a clinically safe dose of EPO is therefore necessary before clinical trials can be considered. Aiming for clinical progress and feasibility, the present study was carried out with a view to evaluating the efficacy of a single, low-dose EPO to stimulate bone healing in a large-animal study. The dose of EPO was chosen based on the following considerations. The translation of the minimally effective dose in cell studies into large-animal models is difficult (Rölfing et al. 2013). The rather low dosage of 2,700 IU/animal, equivalent to 18.5 ± 2.0 IU/kg, was chosen in order to minimize the systemic effect of EPO due to safety concerns and in order to minimize the potential effect on the within-subject controls. In anemic patients, 20–240 IU/kg are injected subcutaneously or intravenously 3 times a week. The hypothesis was that 900 IU site-specifically applied EPO would increase bony ingrowth compared to a saline-treated control in a porcine calvarial defect model.