Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear.In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary – and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies.All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation.It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.     

Dimerization of SLX4 contributes to functioning of the SLX4-nuclease complex

The Fanconi anemia protein SLX4 assembles a genome and telomere maintenance toolkit, consisting of the nucleases SLX1, MUS81 and XPF. Although it is known that SLX4 acts as a scaffold for building this complex, the molecular basis underlying this function of SLX4 remains unclear. Here, we report that functioning of SLX4 is dependent on its dimerization via an oligomerization motif called the BTB domain. We solved the crystal structure of the SLX4_BTB dimer, identifying key contacts (F681 and F708) that mediate dimerization. Disruption of BTB dimerization abrogates nuclear foci formation and telomeric localization of not only SLX4 but also of its associated nucleases. Furthermore, dimerization-deficient SLX4 mutants cause defective cellular response to DNA interstrand crosslinking agent and telomere maintenance, underscoring the contribution of BTB domain-mediated dimerization of SLX4 in genome and telomere maintenance.     

Biochemistry and haematology values for the baboon (Papio hamadryas): the effects of sex, growth, development and age.

A retrospective study evaluated the influence of sex and age on plasma biochemistry and haematology parameters in a captive-bred colony of baboons. Over 1,140 ETDA and heparin blood samples were obtained from 160 clinically normal baboons between the ages of 11 months and 11 years. Data for these blood tests were analysed for the effects of sex, age and sex age interactions. Sex, age and sex age interactions were detected for many plasma biochemistry and haematological parameters. The reference range values for platelets, white-blood cells and mean corpuscular volume and plasma chloride, glucose, total protein and iron were higher (P < 0.01) and red blood cell, plasma sodium, potassium, total CO2, creatinine, urea, total bilirubin, albumin, alkaline phosphate, gamma glutamyl transpeptidase and phosphate were lower (P < 0.01) in the female compared to the male population. Sex age interactions (P < 0.05) were seen with haemoglobin, white blood cells, haematocrit, mean corpuscular volume, sodium, creatinine, urea, calcium, phosphate, total bilirubin, total protein alkaline phosphatase, the liver enzymes and triglycerides. Plasma alkaline phosphatase was highest ( > 800 micro/l) in young juveniles of both sexes; creatinine was higher in older ( > 4 years) compared to younger baboons of the same sex (P < 0.05). Plasma cholesterol and triglycerides were greater (P < 0.01) in young baboons compared to older animals.     

Measurement of pO2 by luminescence lifetime spectroscopy: A comparative study of the phototoxicity and sensitivity of [Ru(Phen)3]2+ and PdTCPP in vivo

Dysfunctions in tissue metabolism can be detected at early stages by oxygen partial pressure (pO₂) measurement. The measurement of emission lifetimes offers very promising and non‐invasive approach to estimate pO₂ in vivo. This study compares two extensively used oxygen sensors and assesses their in vivo oxygen sensitivity and phototoxic effect. Luminescence lifetime of Ru‐polypyridyl complex and of Pd‐porphyrin is measured in the Chick's Chorioallantoic Membrane (CAM) model with a dedicated optical fiber‐based, time‐resolved spectrometer. The Pd‐porphyrin luminescence lifetimes measured in the CAM model exposed to different pO₂ levels are longer and have a broader dynamic range (10–100 μs) than those of Ru‐polypyridyl complex (0.6–1 μs). The combined statistical analysis based on an estimate of the kurtosis and skewness, bootstrapping method and routine normality tests is performed. The indicators of the averages and signal to noise ratio stability are also calculated. The combination of several data processing allows selection of the better sensor for a given application. In particular, it is found that the advantage of Ru‐polypyridyl complex over Pd‐porphyrin is two‐fold: i) Ru‐polypyridyl complex datasets have consistently better statistical characteristics, ii) Ru‐polypyridyl exhibits lower cytotoxicity.

     

Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma

Background

There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.

Methods and Findings

Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).

Conclusions

Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.Trial registration: http://www.ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT00047073","term_id":"NCT00047073"}}NCT00047073).     

gamma-Tocopherol nebulization by a lipid aerosolization device improves pulmonary function in sheep with burn and smoke inhalation injury

Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.     

Phage response to CRISPR-encoded resistance in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.