Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries

Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs), many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro, binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits, which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.     

A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors

Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment.KEY WORDS: Glioblastoma, Mouse model, PI3K and MEK inhibition, Apoptosis     

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Use of ESI-FTICR-MS to Characterize Dissolved Organic Matter in Headwater Streams Draining Forest-Dominated and Pasture-Dominated Watersheds

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) has proven to be a powerful technique revealing complexity and diversity of natural DOM molecules, but its application to DOM analysis in grazing-impacted agricultural systems remains scarce. In the present study, we presented a case study of using ESI-FTICR-MS in analyzing DOM from four headwater streams draining forest- or pasture-dominated watersheds in Virginia, USA. In all samples, most formulas were CHO compounds (71.8–87.9%), with other molecular series (CHOS, CHON, CHONS, and CHOP (N, S)) accounting for only minor fractions. All samples were dominated by molecules falling in the lignin-like region (H/C = 0.7–1.5, O/C = 0.1–0.67), suggesting the predominance of allochthonous, terrestrial plant-derived DOM. Relative to the two pasture streams, DOM formulas in the two forest streams were more similar, based on Jaccard similarity coefficients and nonmetric multidimensional scaling calculated from Bray-Curtis distance. Formulas from the pasture streams were characterized by lower proportions of aromatic formulas and lower unsaturation, suggesting that the allochthonous versus autochthonous contributions of organic matter to streams were modified by pasture land use. The number of condensed aromatic structures (CAS) was higher for the forest streams, which is possibly due to the controlled burning in the forest-dominated watersheds and suggests that black carbon was mobilized from soils to streams. During 15-day biodegradation experiments, DOM from the two pasture streams was altered to a greater extent than DOM from the forest streams, with formulas with H/C and O/C ranges similar to protein (H/C = 1.5–2.2, O/C = 0.3–0.67), lipid (H/C = 1.5–2.0, O/C = 0–0.3), and unsaturated hydrocarbon (H/C = 0.7–1.5, O/C = 0–0.1) being the most bioreactive groups. Aromatic compound formulas including CAS were preferentially removed during combined light+bacterial incubations, supporting the contention that black carbon is labile to light alterations. Collectively, our data demonstrate that headwater DOM composition contains integrative information on watershed sources and processes, and the application of ESI-FTICR-MS technique offers additional insights into compound composition and reactivity unrevealed by fluorescence and stable carbon isotopic measurements.     

Atypical systemic leishmaniasis to be considered in the differential of patients presenting with depressed immunity

Background

Systemic leishmaniasis has been known to present with prolonged fever, hepatosplenomegaly and wasting. Beside this classical form, a sub-clinical form has been identified. It is described with either one or two of the above symptoms missing; other findings have been reported instead, such as lymphadenopathy and anemia. In this report, we reveal a third unsuspected form which we are referring to as “atypical”.

Methodology/Principal Findings

Patients suspected to be immune-deficient were referred to our immunology specialized laboratory to study some aspects of their immune functions (not normally covered in the general laboratory). Multiple specialized tests were performed, including microscopic examinations using appropriate stains, and mainly cultures of biopsies on several types of specialized media. 19·4% of 160 patients were found to have close to normal laboratory profiles, but exhibited dysfunctional macrophages laden with Leishmania parasites.

Conclusions/Significance

Findings such as the ones we obtained allowed us to uncover the presence of patients with an atypical form of systemic leishmaniasis. It presents with symptoms masquarading a condition in which the immune system is non functional. This predisposes patients to recurrent secondary infections resulting in clinical pictures with a great variety of signs and symptoms. These findings alerted us to the fact that systemic leishmaniasis presents with a much wider spectrum of signs and symptoms than so far suspected and is far more common than diagnosed to date. Furthermore, among these 31 patients was a number of adults. This proved that in our area systemic leishmaniasis is surely not limited to the pediatric age group. Our recommendation is to entertain the diagnosis of atypical systemic leishmaniasis in any patient with an unexplained depressed immunity state and in whom no obvious immunologic defect can be identified.     

Cytokines in response to proteins predicted in genomic regions of difference of Mycobacterium tuberculosis

Cellular immune responses are responsible for both protection and pathogenesis in tuberculosis, and are mediated/regulated by a complex network of pro-inflammatory, T helper (Th) type 1 and type 2 cytokines. In this study, the secretion of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8 and IL-1β; Th1 cytokines interferon-gamma (IFN-γ), IL-2 and tumor necrosis factor-beta (TNF-β); and Th2 cytokines IL-4, IL-5 and IL-10 by the peripheral blood mononuclear cells (PBMCs) of pulmonary tuberculosis patients was studied. PBMCs were cultured in vitro in the absence and presence of complex mycobacterial antigens and peptides corresponding to 11 regions of difference (RD) of Mycobacterium tuberculosis that are deleted/absent in all vaccine strains of Mycobacterium bovis bacillus Calmette-Guérin (BCG). The culture supernatants were tested for secreted cytokines by FlowCytomix assay. PBMCs from the majority of patients (53-100%) spontaneously secreted detectable concentrations of all cytokines tested, except for IL2 (29%) and IL-10 (41%). The profiles of proinflammatory cytokines were largely similar for various complex antigens or RD peptides. However, with respect to Th1 and Th2 cytokines, the antigens could be divided into three groups; the first with Th1-bias (culture filtrate of M. tuberculosis, RD1, RD5, RD7, RD9 and RD10), the second with Th2-bias (whole cells and cell walls of M. tuberculosis, RD12, RD13 and RD15), and the third without Th1/Th2-bias (M. bovis BCG, RD4, RD6 and RD11). Complex mycobacterial antigens and RD proteins with Th1- and Th2-biases may have roles in protection and pathogenesis of tuberculosis, respectively.     

Actin-filament stochastic dynamics mediated by ADF/cofilin

BACKGROUND: The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, we combined evanescent-wave microscopy with a biomimetic system and followed the behavior of single actin filaments in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. RESULTS: In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. CONCLUSIONS: Our proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells.     

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).     

Pharmacological glycerol-3-phosphate acyltransferase inhibition decreases food intake and adiposity and increases insulin sensitivity in diet-induced obesity

Storage of excess calories as triglycerides is central to obesity and its associated disorders. Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial step in acylglyceride syntheses, including triglyceride synthesis. We utilized a novel small-molecule GPAT inhibitor, FSG67, to investigate metabolic consequences of systemic pharmacological GPAT inhibition in lean and diet-induced obese (DIO) mice. FSG67 administered intraperitoneally decreased body weight and energy intake, without producing conditioned taste aversion. Daily FSG67 (5 mg/kg, 15.3 μmol/kg) produced gradual 12% weight loss in DIO mice beyond that due to transient 9- to 10-day hypophagia (6% weight loss in pair-fed controls). Continued FSG67 maintained the weight loss despite return to baseline energy intake. Weight was lost specifically from fat mass. Indirect calorimetry showed partial protection by FSG67 against decreased rates of oxygen consumption seen with hypophagia. Despite low respiratory exchange ratio due to a high-fat diet, FSG67-treated mice showed further decreased respiratory exchange ratio, beyond pair-fed controls, indicating enhanced fat oxidation. Chronic FSG67 increased glucose tolerance and insulin sensitivity in DIO mice. Chronic FSG67 decreased gene expression for lipogenic enzymes in white adipose tissue and liver and decreased lipid accumulation in white adipose, brown adipose, and liver tissues without signs of damage. RT-PCR showed decreased gene expression for orexigenic hypothalamic neuropeptides AgRP or NPY after acute and chronic systemic FSG67. FSG67 given intracerebroventricularly (100 and 320 nmol icv) produced 24-h weight loss and feeding suppression, indicating contributions from direct central nervous system sites of action. Together, these data point to GPAT as a new potential therapeutic target for the management of obesity and its comorbidities.