Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure–function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in ‘designer cellulosomes’. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

THetA: inferring intra-tumor heterogeneity from high-throughput DNA sequencing data

Tumor samples are typically heterogeneous, containing admixture by normal, non-cancerous cells and one or more subpopulations of cancerous cells. Whole-genome sequencing of a tumor sample yields reads from this mixture, but does not directly reveal the cell of origin for each read. We introduce THetA (Tumor Heterogeneity Analysis), an algorithm that infers the most likely collection of genomes and their proportions in a sample, for the case where copy number aberrations distinguish subpopulations. THetA successfully estimates normal admixture and recovers clonal and subclonal copy number aberrations in real and simulated sequencing data. THetA is available at http://compbio.cs.brown.edu/software/.     

Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation

Background

Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients.

Methods and Findings

Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women''s Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6–15.8, p = 1×10⁻⁷) and ME ARDS (OR 8.4, 95% CI 2.9–24.2, p = 9×10⁻⁵) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10⁻⁴) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers.

Conclusions

Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients. Please see later in the article for the Editors'' Summary     

Glucose-6-phosphate isomerase deficiency results in mTOR activation, failed translocation of lipin 1α to the nucleus and hypersensitivity to glucose: Implications for the inherited glycolytic disease

Inherited glucose-6-phosphate isomerase (GPI) deficiency is the second most frequent glycolytic erythroenzymopathy in humans. Patients present with non-spherocytic anemia of variable severity and with neuromuscular dysfunction. We previously described Chinese hamster (CHO) cell lines with mutations in GPI and loss of GPI activity. This resulted in a temperature sensitivity and severe reduction in the synthesis of glycerolipids due to a reduction in phosphatidate phosphatase (PAP). In the current article we attempt to describe the nature of this pleiotropic effect. We cloned and sequenced the CHO lipin 1 cDNA, a gene that codes for PAP activity. Overexpression of lipin 1 in the GPI-deficient cell line, GroD1 resulted in increased PAP activity, however it failed to restore glycerolipid biosynthesis. Fluorescence microscopy showed a failure of GPI-deficient cells to localize lipin 1α to the nucleus. We also found that glucose-6-phosphate levels in GroD1 cells were 10-fold over normal. Lowering glucose levels in the growth medium partially restored glycerolipid biosynthesis and nuclear localization of lipin 1α. Western blot analysis of the elements within the mTOR pathway, which influences lipin 1 activity, was consistent with an abnormal activation of this system. Combined, these data suggest that GPI deficiency results in an accumulation of glucose-6-phosphate, and possibly other glucose-derived metabolites, leading to activation of mTOR and sequestration of lipin 1 to the cytosol, preventing its proper functioning. These results shed light on the mechanism underlying the pathologies associated with inherited GPI deficiency and the variability in the severity of the symptoms observed in these patients.     

Eosinophils as a novel cell source of prostaglandin D2: autocrine role in allergic inflammation

PGD(2) is a key mediator of allergic inflammatory diseases that is mainly synthesized by mast cells, which constitutively express high levels of the terminal enzyme involved in PGD(2) synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD(2). PGD(2) synthesis was evaluated within human blood eosinophils, in vitro differentiated mouse eosinophils, and eosinophils infiltrating inflammatory site of mouse allergic reaction. Biological function of eosinophil-derived PGD(2) was studied by employing inhibitors of synthesis and activity. Constitutive expression of H-PGDS was found within nonstimulated human circulating eosinophils. Acute stimulation of human eosinophils with A23187 (0.1-5 μM) evoked PGD(2) synthesis, which was located at the nuclear envelope and was inhibited by pretreatment with HQL-79 (10 μM), a specific H-PGDS inhibitor. Prestimulation of human eosinophils with arachidonic acid (10 μM) or human eotaxin (6 nM) also enhanced HQL-79-sensitive PGD(2) synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C(4) synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD(2) in response to arachidonic acid stimulation. In vivo, at late time point of the allergic reaction, infiltrating eosinophils found at the inflammatory site appeared as an auxiliary PGD(2)-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD(2), hence representing during allergic inflammation an extra cell source of PGD(2), which functions as an autocrine signal for eosinophil activation.     

Effects on sleep and dopamine levels of microdialysis perfusion of cannabidiol into the lateral hypothalamus of rats

AimsThe major non-psychoactive component of Cannabis sativa, cannabidiol (CBD), displays a plethora of actions including wakefulness. In the present study, we addressed whether perfusing CBD via microdialysis into lateral hypothalamus (LH) during the lights-on period would modify the sleep-wake cycle of rats as well as the contents of dopamine (DA) collected from nucleus accumbens (AcbC). Additionally, we tested whether perfusion of CBD into LH would block the sleep rebound after a sleep deprivation period.Main methodsElectroencephalogram and electromyogram electrodes were implanted in rats as well as a guide-cannula aimed to LH or AcbC. CBD perfusion was carried out via cannulae placed into LH whereas contents of DA were collected from AcbC and analyzed using HPLC means.Key findingsWe found that microdialysis perfusion of CBD (30, 60, or 90 nM) into LH of rat enhances alertness and suppresses sleep. This effect was accompanied with an increase in DA extracellular levels collected from the AcbC. Furthermore, perfusion of CBD into LH after total sleep deprivation prevented the sleep rebound.SignificanceThese findings enhance the investigation about the neurobiological properties of CBD on sleep modulation.     

Extended-spectrum Beta-lactamase gene sequences in gram-negative saprophytes on retail organic and nonorganic spinach

A substantial proportion of infections caused by drug-resistant Gram-negative bacteria (GNB) in community and health care settings are recognized to be caused by evolutionarily related GNB strains. Their global spread has been suggested to occur due to human activities, such as food trade and travel. These multidrug-resistant GNB pathogens often harbor mobile drug resistance genes that are highly conserved in their sequences. Because they appear across different GNB species, these genes may have origins other than human pathogens. We hypothesized that saprophytes in common human food products may serve as a reservoir for such genes. Between July 2007 and April 2008, we examined 25 batches of prepackaged retail spinach for cultivatable GNB population structure by 16S rRNA gene sequencing and for antimicrobial drug susceptibility testing and the presence of extended-spectrum beta-lactamase (ESBL) genes. We found 20 recognized GNB species among 165 (71%) of 231 randomly selected colonies cultured from spinach. Twelve strains suspected to express ESBLs based on resistance to cefotaxime and ceftazidime were further examined for bla(CTX-M) and bla(TEM) genes. We found a 712-bp sequence in Pseudomonas teessidea that was 100% identical to positions 10 to 722 of an 876-bp bla(CTX-M-15) gene of an E. coli strain. Additionally, we identified newly recognized ESBL bla(RAHN-2) sequences from Rahnella aquatilis. These observations demonstrate that saprophytes in common fresh produce can harbor drug resistance genes that are also found in internationally circulating strains of GNB pathogens; such a source may thus serve as a reservoir for drug resistance genes that ultimately enter pathogens to affect human health.     

Deuterium reveals the dynamics of notch activation

Notch activation requires unfolding of a juxtamembrane negative regulatory domain (NRR). Tiyanont et?al. (2011) analyzed the dynamics of NRR unfolding in the presence of EGTA. As predicted from the crystal structure and deletion analyses, the lin-Notch repeats unfold first, facilitating access by ADAM proteases. Surprisingly, the heterodimerization domain remains stable.     

Parahippocampal cortex: translating vision into space

Two recent imaging studies have shed new light on information representation in human parahippocampal cortex. Despite their different approaches, the two studies both support the view that this brain region represents space at an elementary level.     

How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation

Two critical cysteine residues in the copper-A site (Cu_A) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu²⁺ transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E′₀ = −231 mV) as compared with that of TlpA (E′₀ = −256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10⁴ m⁻¹ s⁻¹ that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu_A-CoxB complex may be bypassed in vivo by high environmental Cu²⁺ concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.