Olfactory Bulbectomy Leads to the Development of Epilepsy in Mice

There is a clear link between epilepsy and depression. Clinical data demonstrate a 30–35% lifetime prevalence of depression in patients with epilepsy, and patients diagnosed with depression have a three to sevenfold higher risk of developing epilepsy. Traditional epilepsy models partially replicate the clinical observations, with the demonstration of depressive traits in epileptic animals. Studies assessing pro-epileptogenic changes in models of depression, however, are more limited. Here, we examined whether a traditional rodent depression model—bilateral olfactory bulbectomy—predisposes the animals towards the development of epilepsy. Past studies have demonstrated increased neuronal excitability after bulbectomy, but continuous seizure monitoring had not been conducted. For the present study, we monitored control and bulbectomized animals by video-EEG 24/7 for approximately two weeks following the surgery to determine whether they develop spontaneous seizures. All seven bulbectomized mice exhibited seizures during the monitoring period. Seizures began about one week after surgery, and occurred in clusters with severity increasing over the monitoring period. These results suggest that olfactory bulbectomy could be a useful model of TBI-induced epilepsy, with advantages of relatively rapid seizure onset and a high number of individuals developing the disease. The model may also be useful for investigating the mechanisms underlying the bidirectional relationship between epilepsy and depression.     

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl^-release in intestinal cells, including the human colonic carcinoma T₈₄ cell line, involving increased cGMP and membrane alkalization due to reduced Na⁺/H⁺ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pH_i), and NHE1, NHE2, and NHE4 are expressed in T₈₄ cells, we characterized the STa role as modulator of these exchangers. pH_i was assayed by the NH₄Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pH_i recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H⁺ efflux (J _H ⁺) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J _H ⁺ (~63%), without altering basal pH_i (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pH_i units. The dpHi/dt and J _H ⁺ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and J _H ⁺ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T₈₄ cells with STa results in reduced NHE4 activity leading to a lower capacity of pH_i recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.     

Acute Dietary Nitrate Supplementation and Exercise Performance in COPD: A Double-Blind,Placebo-Controlled,Randomised Controlled Pilot Study

Background

Dietary nitrate supplementation can enhance exercise performance in healthy people, but it is not clear if it is beneficial in COPD. We investigated the hypotheses that acute nitrate dosing would improve exercise performance and reduce the oxygen cost of submaximal exercise in people with COPD.

Methods

We performed a double-blind, placebo-controlled, cross-over single dose study. Subjects were randomised to consume either nitrate-rich beetroot juice (containing 12.9mmoles nitrate) or placebo (nitrate-depleted beetroot juice) 3 hours prior to endurance cycle ergometry, performed at 70% of maximal workload assessed by a prior incremental exercise test. After a minimum washout period of 7 days the protocol was repeated with the crossover beverage.

Results

21 subjects successfully completed the study (age 68±7years; BMI 25.2±5.5kg/m²; FEV₁ percentage predicted 50.1±21.6%; peak VO₂ 18.0±5.9ml/min/kg). Resting diastolic blood pressure fell significantly with nitrate supplementation compared to placebo (-7±8mmHg nitrate vs. -1±8mmHg placebo; p = 0.008). Median endurance time did not differ significantly; nitrate 5.65 (3.90–10.40) minutes vs. placebo 6.40 (4.01–9.67) minutes (p = 0.50). However, isotime oxygen consumption (VO₂) was lower following nitrate supplementation (16.6±6.0ml/min/kg nitrate vs. 17.2±6.0ml/min/kg placebo; p = 0.043), and consequently nitrate supplementation caused a significant lowering of the amplitude of the VO₂-percentage isotime curve.

Conclusions

Acute administration of oral nitrate did not enhance endurance exercise performance; however the observation that beetroot juice caused reduced oxygen consumption at isotime suggests that further investigation of this treatment approach is warranted, perhaps targeting a more hypoxic phenotype.

Trial Registration

ISRCTN Registry ISRCTN66099139     

Are bird density,species richness and community structure similar between native woodlands and non-native plantations in an area with a generalist bird fauna?

This study compared the bird assemblages of native semi-natural woodlands and non-native Sitka spruce (Picea sitchensis) plantations in Ireland to identify what vegetation variables most influenced birds and to identify management targets in plantations to maximise future bird conservation. Point counts were conducted in 10 Oak (Quercus spp.) and 10 Ash (Fraxinus excelsior) native woodlands and in five Mid-rotation (20–30 years old) and five Mature (30–50 years old) Sitka spruce plantations. Ordination was used to characterise woodland types according to their constituent bird species. Total bird density (calculated using Distance software) and species richness were assessed for the different woodland types. Oak and Ash woodland bird assemblages were separated from Mid-rotation and Mature plantations by the ordination. There was no difference in total bird density between any of the woodland types. Oak woodlands had significantly higher species richness than either Mid-rotation or Mature Sitka spruce plantations. Ash had higher species richness than Mature Sitka spruce plantations. Understorey vegetation was negatively associated with total bird density, which also varied with survey year. Understorey vegetation was positively associated with species richness. Reasons for the relationships between vegetation and bird assemblages are discussed. Management should seek to increase shrub and understorey vegetation in the Mid-rotation phase to improve the contribution of plantations to bird conservation.     

Label-Free Bacterial Imaging with Deep-UV-Laser-Induced Native Fluorescence

We introduce a near-real-time optical imaging method that works via the detection of the intrinsic fluorescence of life forms upon excitation by deep-UV (DUV) illumination. A DUV (<250-nm) source enables the detection of microbes in their native state on natural materials, avoiding background autofluorescence and without the need for fluorescent dyes or tags. We demonstrate that DUV-laser-induced native fluorescence can detect bacteria on opaque surfaces at spatial scales ranging from tens of centimeters to micrometers and from communities to single cells. Given exposure times of 100 μs and low excitation intensities, this technique enables rapid imaging of bacterial communities and cells without irreversible sample alteration or destruction. We also demonstrate the first noninvasive detection of bacteria on in situ-incubated environmental experimental samples from the deep ocean (Lo''ihi Seamount), showing the use of DUV native fluorescence for in situ detection in the deep biosphere and other nutrient-limited environments.Bacteria are widely recognized for living in extreme environments and as integral players in processes as varied as weathering, corrosion, environmental remediation, pathogenesis, and symbiosis (3, 4, 26). In most of these cases, surface-bound bacteria play key roles (1, 7, 19) and pose a particular challenge for researchers: the detection and imaging of life on reflective and/or fluorescent surfaces at the microbial (μm) scale (5, 12, 18). In environments ranging from the deep subsurface biosphere, dry deserts, and deep ice cores to hospitals and clean rooms, concentrations of bacteria, either as spores or active cells, can range from 10⁹ to less than 1,000 cells/gram (14, 22, 24, 25, 29, 34). Finding and quantifying these microbes when they are on surfaces usually involves epifluorescence techniques, using dyes that bind to DNA or proteins, and examining the fluorescence of those dyes under UV or visible illumination (6, 8, 9, 16, 23, 31).Current tagging methods offer a number of significant disadvantages. First, the mineral surfaces on which the microbes are found are often themselves highly fluorescent, making the microbes difficult or impossible to differentiate; second, the act of adding the fluorescent probe can alter the physical and chemical nature of the system; additionally, nonspecific binding can lead to overestimation of cell abundance (2, 18). Because of the problems associated with the fluorescence of minerals and staining to detect microbial cells, researchers typically resort to physically removing cells from surfaces and staining/counting them separately from their matrix (12). This is an inefficient process that involves both cell loss and the loss of information about the mineralogical context that may have an influence on the microbial ecology. More recently, cell staining of active cells with SYBR green 1 and a computer-assisted analysis method has demonstrated an ability to separate fluorescent cells from nonspecific binding (17). However, a label-free method to search for and quantify the distribution and abundance of bacteria on natural samples over multiple spatial scales has not been available.Label-free optical approaches using Raman scattering methods have been offered as a nondestructive imaging solution (13, 27). However, these systems utilize laser energies greater than 1 × 10⁹ joules/cm², exceeding the energies necessary for chemical damage to the cell (33), require relatively flat surfaces for optimal collection efficiency, and can suffer from background fluorescence of the target and the substrate it may reside on.In response to these challenges, we have developed an optical method that enables detection and imaging of single bacterial cells on natural and opaque surfaces and assessment of bacterial density and distribution of single cells to biofilms over spatial scales ranging from microns to centimeters. The method utilizes deep-UV (DUV) (<250-nm)-laser-induced native fluorescence of organic components intrinsic to the cell or spore while avoiding autofluorescence interference from the substrate. Here we show DUV native fluorescence as a near-real-time optical imaging method and demonstrate the first noninvasive detection of bacteria on in situ-incubated environmental experimental samples from the deep ocean (Lo''ihi Seamount) for which we correlate the bacterial biomass to distributions of the iron-oxide precipitates.     

Non-β-cell progenitors of β-cells in pregnant mice

Pregnancy is a normal physiological condition in which the maternal β-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of β-cells to analyse the origin of new β-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, β-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HP AP). First, we conclude that the lineage tracing system was highly specific for β-cells. Secondly, we scored the proportion of the β-cells marked with HP AP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labeling index in pregnant animal pancreata, compared to nonpregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labeling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-β-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only β-cell duplication, but also the activation of a non-β-cell progenitor population. Further, there was no transdifferentiation of β-cells to other cell types in a two and half month period following labeling, including the period of pregnancy.