A P-loop Mutation in G�� Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gα_i1(G42R) binding to GDP·AlF₄ ⁻ or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gα_q(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.     

Photodimerization in pyrimidine-substituted dipeptides.

Ten N(epsilon)-glycylornithineamide derivatives have been synthesized containing various N(alpha)-linked pyrimidine-1-ylacetyl groups which can undergo (2pi + 2pi) photodimerization on irradiation with UV light at 254 nm. The dimerization efficiency of the free and bound pyrimidine groups was compared in aqueous solution: it was dependent on the substitution of the pyrimidine ring. N(alpha),N(alpha')-bis-(uracil-1-ylacetyl)-(N(epsilon)-glycylornithineamide) and N(alpha),N(alpha')-bis-(5-bromouracil-1-ylacetyl)-(N(epsilon)-glycylornithineamide) were identified as possible candidates for optical data storage.     

Pulses of darkness shift the phase of a circadian rhythm in an insectivorous bat

Summary Microspectrophotometric measurements on the retina ofDeilephila elpenor were performed using fresh (cut by razor blade) as well as frozen (cryostate microtome) sections. Difference spectra from various parts of the rhabdom clearly showed that in the distal part of the rhabdom the UV pigment is predominant, while in the medial part only green pigment was found. From the difference spectra, wave-length and relative height of the absorption maxima of rhodopsins and metarhodopsins were computed and were found to be in close agreement with other results reported in the literature. These findings confirm the work of Welsch (1977) who concluded from his electron microscopic analysis that the two distal receptor cells are UV sensitive, while the six median receptor cells are green sensitive. There is also good evidence to assume that the ninth, proximal receptor is violet sensitive.We would like to thank Mrs. E. Brüning for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft SFB 114 Bionach.     

Optical Spectral Surveillance of Breast Tissue Landscapes for Detection of Residual Disease in Breast Tumor Margins

We demonstrate a strategy to “sense” the micro-morphology of a breast tumor margin over a wide field of view by creating quantitative hyperspectral maps of the tissue optical properties (absorption and scattering), where each voxel can be deconstructed to provide information on the underlying histology. Information about the underlying tissue histology is encoded in the quantitative spectral information (in the visible wavelength range), and residual carcinoma is detected as a shift in the histological landscape to one with less fat and higher glandular content. To demonstrate this strategy, fully intact, fresh lumpectomy specimens (n = 88) from 70 patients were imaged intra-operatively. The ability of spectral imaging to sense changes in histology over large imaging areas was determined using inter-patient mammographic breast density (MBD) variation in cancer-free tissues as a model system. We discovered that increased MBD was associated with higher baseline β-carotene concentrations (p = 0.066) and higher scattering coefficients (p = 0.007) as measured by spectral imaging, and a trend toward decreased adipocyte size and increased adipocyte density as measured by histological examination in BMI-matched patients. The ability of spectral imaging to detect cancer intra-operatively was demonstrated when MBD-specific breast characteristics were considered. Specifically, the ratio of β-carotene concentration to the light scattering coefficient can report on the relative amount of fat to glandular density at the tissue surface to determine positive margin status, when baseline differences in these parameters between patients with low and high MBD are taken into account by the appropriate selection of threshold values. When MBD was included as a variable a priori, the device was estimated to have a sensitivity of 74% and a specificity of 86% in detecting close or positive margins, regardless of tumor type. Superior performance was demonstrated in high MBD tissue, a population that typically has a higher percentage of involved margins.     

Studies on SAGO wastewater treatment using anaerobic rotating biological contactor

Experimental studies were done in a laboratory scale Anaerobic Rotating Biological Contactor (RBC), for treatment of Synthetic sago wastewater. This paper describes the development and laboratory testing of an Anaerobic RBC process that couples the advantages of the fixed film horizontal flow RBC process with the high strength, starch degradation capabilities of anaerobic systems. The reactor was operated at ambient temperature and was subjected to organic and hydraulic loading rates. The reactor performance with respect to Chemical Oxygen Demand (COD) removal, alkalinity, volatile acids at each stage and biogas production were evaluated. The Anaerobic RBC reactor liquid volume is 70 litres and total disc surface area is 4.45 m². The reactor was operated with about 100% of the disc area submerged and with a rotational speed held constant at 9?rev/min. The synthetic sago wastewater was started with a COD value of 1087?mg/l at a hydraulic retention time(HRT) of 42?h and it was varied till maximum COD of 9522?mg/l. From the present study, the optimum COD load was found to be 6860?mg/l with a COD removal efficiency of 97.2%.With this optimum COD load, hydraulic loading rate(HLR) study was done at 24?h to 48?h HRT. COD removal efficiencies at hydraulic loading rates were compared with the work of Subrahmanyam &; Sastry (1988). From the present study, the proportionality coefficient was found to be 1.18 with process efficiencies at different hydraulic loading rates.     

Treatment of distillery spentwash by hybrid UASB reactor

A laboratory-scale hybrid UASB reactor, which combined an UASB in the lower part and a filter in the upper part, was used for the treatment of distillery spentwash. The reactor was operated under ambient conditions for 380 days. Using anaerobically digested sewage sludge as a seed, the start-up of the reactor and the cultivation of active granular sludge was completed within three months period. Scanning electron microscopic (SEM) observation of the granules showed the presence of Mehtanonthrix-like bacteria as the dominant species. Following the start-up the organic loading rate (OLR) was increased, stepwise, to 36 kg COD/m³ · d at a constant hydraulic retention time (HRT) of 6 h. COD removal efficiency was 80% even at a high OLR of 36 kg COD/m³ · d. Biogas rich in methane content (80%), with a maximum specific biogas yield of 0.40 m³ CH₄/kg · COD was produced. Polypropylene pall rings filter medium in the upper-third of the reactor was very effective as a gas-liquid-solid (GLS) separator, and retained the biomass in addition. The study indicated that hybrid UASB is a very feasible alternative for the treatment of high-strength wastewaters like distillery spentwash.     

Antibiofilm Activity of Biosurfactant Producing Coral Associated Bacteria Isolated from Gulf of Mannar

Coral Associated Bacteria (CAB) (N = 22) isolated from the mucus of the coral Acropora digitifera were screened for biosurfactants using classical screening methods; hemolysis test, lipase production, oil displacement, drop collapse test and emulsifying activity. Six CAB (U7, U9, U10, U13, U14, and U16) were found to produce biosurfactants and were identified by 16S ribosomal RNA gene sequencing as Providencia rettgeri, Psychrobacter sp., Bacillus flexus, Bacillus anthracis, Psychrobacter sp., and Bacillus pumilus respectively. Their cell surface hydrophobicity was determined by Microbial adhesion to hydrocarbon assay and the biosurfactants produced were extracted and characterized by Fourier Transform Infrared spectroscopy. Since the biosurfactants are known for their surface modifying capabilities, antibiofilm activity of positive isolates was evaluated against biofilm forming Pseudomonas aeruginosa ATCC10145. Stability of the active principle exhibiting antibiofilm activity was tested through various temperature treatments ranging from 60 to 100 °C and Proteinase K treatment. CAB isolates U7 and U9 exhibited stable antibiofilm activity even after exposure to higher temperatures which is promising for the development of novel antifouling agents for diverse industrial applications. Further, this is the first report on biosurfactant production by a coral symbiont.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0474-8) contains supplementary material, which is available to authorized users.     

IFN-α confers resistance of systemic lupus erythematosus nephritis to therapy in NZB/W F1 mice

The critical role of IFN-α in the pathogenesis of human systemic lupus erythematosus has been highlighted in recent years. Exposure of young lupus-prone NZB/W F1 mice to IFN-α in vivo leads to an accelerated lupus phenotype that is dependent on T cells and is associated with elevated serum levels of BAFF, IL-6, and TNF-α, increased splenic expression of IL-6 and IL-21, formation of large germinal centers, and the generation of large numbers of short-lived plasma cells that produce IgG2a and IgG3 autoantibodies. In this study, we show that both IgG2a and IgG3 autoantibodies are pathogenic in IFN-α-accelerated lupus, and their production can be dissociated by using low-dose CTLA4-Ig. Only high-dose CTLA4-Ig attenuates both IgG2a and IgG3 autoantibody production and significantly delays death from lupus nephritis. In contrast, BAFF/APRIL blockade has no effect on germinal centers or the production of IgG anti-dsDNA Abs but, if given at the time of IFN-α challenge, delays the progression of lupus by attenuating systemic and renal inflammation. Temporary remission of nephritis induced by combination therapy with cyclophosphamide, anti-CD40L Ab, and CTLA4-Ig is associated with the abrogation of germinal centers and depletion of short-lived plasma cells, but relapse occurs more rapidly than in conventional NZB/W F1 mice. This study demonstrates that IFN-α renders NZB/W F1 relatively resistant to therapeutic intervention and suggests that the IFN signature should be considered when randomizing patients into groups and analyzing the results of human clinical trials in systemic lupus erythematosus.     

Phenol, 2,4-bis(1,1-dimethylethyl) of marine bacterial origin inhibits quorum sensing mediated biofilm formation in the uropathogen Serratia marcescens

Intercellular communication in bacteria (quorum sensing, QS) is an important phenomenon in disease dissemination and pathogenesis, which controls biofilm formation also. This study reports the anti-QS and anti-biofilm efficacy of seaweed Gracilaria gracilis associated Vibrio alginolyticus G16 against Serratia marcescens. Purification and mass spectrometric analysis revealed the active principle as phenol, 2,4-bis(1,1-dimethylethyl) [PD]. PD affected the QS regulated virulence factor production in S. marcescens and resulted in a significant (p < 0.05) reduction in biofilm (85%), protease (41.9%), haemolysin (69.9%), lipase (84.3%), prodigiosin (84.5%) and extracellular polysaccharide (84.62%) secretion without hampering growth, as evidenced by XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. qPCR analysis confirmed the down-regulation of the fimA, fimC, flhD and bsmA genes involved in biofilm formation. Apart from biofilm inhibition and disruption, PD increased the susceptibility of S. marcescens to gentamicin when administered synergistically, which opens another avenue for combinatorial therapy where PD can be used to enhance the efficacy of conventional antibiotics.