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     

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.     

Differential modulation of Alzheimer's disease amyloid beta-peptide accumulation by diverse classes of metal ligands

Biometals have an important role in AD (Alzheimer's disease) and metal ligands have been investigated as potential therapeutic agents for treatment of AD. In recent studies the 8HQ (8-hydroxyquinoline) derivative CQ (clioquinol) has shown promising results in animal models and small clinical trials; however, the actual mode of action in vivo is still being investigated. We previously reported that CQ-metal complexes up-regulated MMP (matrix metalloprotease) activity in vitro by activating PI3K (phosphoinositide 3-kinase) and JNK (c-jun N-terminal kinase), and that the increased MMP activity resulted in enhanced degradation of secreted Abeta (amyloid beta) peptide. In the present study, we have further investigated the biochemical mechanisms by which metal ligands affect Abeta metabolism. To achieve this, we measured the effects of diverse metal ligands on cellular metal uptake and secreted Abeta levels in cell culture. We report that different classes of metal ligands including 8HQ and phenanthroline derivatives and the sulfur compound PDTC (pyrrolidine dithiocarbamate) elevated cellular metal levels (copper and zinc), and resulted in substantial loss of secreted Abeta. Generally, the ability to inhibit Abeta levels correlated with a higher lipid solubility of the ligands and their capacity to increase metal uptake. However, we also identified several ligands that potently inhibited Abeta levels while only inducing minimal change to cellular metal levels. Metal ligands that inhibited Abeta levels [e.g. CQ, 8HQ, NC (neocuproine), 1,10-phenanthroline and PDTC] induced metal-dependent activation of PI3K and JNK, resulting in JNK-mediated up-regulation of metalloprotease activity and subsequent loss of secreted Abeta. The findings in the present study show that diverse metal ligands with high lipid solubility can elevate cellular metal levels resulting in metalloprotease-dependent inhibition of Abeta. Given that a structurally diverse array of ligands was assessed, the results are consistent with the effects being due to metal transport rather than the chelating ligand interacting directly with a receptor.     

Effects of timed administration of doxycycline or methylprednisolone on post-myocardial infarction inflammation and left ventricular remodeling in the rat heart

The development of strategies to ameliorate post-myocardial infarction (MI) remodeling and improve function continues to be an area of clinical importance. Use of steroids for this purpose is controversial since the effects of timed treatment on relevant inflammatory, biochemical and structure/function endpoints are unclear. In a previous report, we demonstrated that use of doxycycline pre-treatment improves post-MI remodeling and passive left ventricular (LV) function. However, the effects of timed doxycyline post-MI treatment are unknown. To examine these issues, we performed a study using a rat MI model. Animals were administered one of the following: doxycycline (DOX), the corticosteroid methylprednisolone (MP), or aqueous vehicle. Treatment was given early, short-term (at time of MI to 24 h post-MI) or late, long term (2–7 days post-MI). Animals were sacrificed at 3, 7 or 42 days post-surgery. We assessed LV hemodynamics, pressure–volume, and pressure–scar strains, histomorphometry, inflammation via measurements of myeloperoxidase activity, and matrix metalloproteinase (MMP) activity. Late MP treatment yielded a robust right-shifted pressure–volume curve, which was accompanied by increased scar strains. Late DOX treatment yielded reduced average heart weight and size and preserved scar thickness. DOX treatment did not suppress inflammation, which contrasts with the suppressive effects of MP. Use of early or late MP yielded increased MMP activity in infarcted and non-infarcted regions. Early and late treatment with DOX yielded infarct–associated MMP activity levels comparable to those of vehicle–treated animals. In conclusion, results indicate that late use of MP yields adverse post-MI structure/function outcomes that correlate with suppression of inflammation and increased MMP activity. These observations contrast with those of DOX, in particular, late treatment where improved outcomes were observed in LV structure and were accompanied by the lack of suppression of inflammation.     

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.     

Synthesis, antifungal and antimicrobial activity of alkylphospholipids

The antifungal, antibacterial and haemolytic activity of a series of alkylphosphocholines (e.g., miltefosine) and alkylglycerophosphocholines (e.g., edelfosine) has been investigated. These compound classes exhibit significant antifungal and moderate antibacterial activities. Several new alkylphosphocholine derivatives with amide or ester bonds in the alkyl chain have been synthesised. These compounds show much lower haemolytic activity than miltefosine. Alkylphosphocholines and alkylglycerophosphocholines show significant promise as novel orally available antifungal and antibacterial therapeutics.