Development of a charcoal paper adenosine triphosphate:pyrophosphate exchange assay: Kinetic characterization of NEDD8 activating enzyme

Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous “E1s” initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [³²P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal powder to separate it from excess [³²P]PPi and then measuring [³²P]ATP in a scintillation counter. We have modified the method by using charcoal paper to capture the nucleotide and a phosphorimager to quantify the [³²P]ATP. The significant increase in throughput that these modifications provide is accomplished without any sacrifice in sensitivity or accuracy compared with the traditional method. To demonstrate this, we reproduce and extend the characterization of the NEDD8 activating enzyme.     

Downscaling Drug Nanosuspension Production: Processing Aspects and Physicochemical Characterization

In this study, scaling down nanosuspension production to 10 mg of drug compound and evaluation of the nanosuspensions to 1 mg of drug compound per test were investigated. Media milling of seven model drug compounds (cinnarizine–indomethacin–itraconazole–loviride–mebendazole–naproxen–phenytoin) was evaluated in a 96-well plate setup (10, 20, and 30 mg) and a glass-vial-based system in a planetary mill (10, 100, and 1,000 mg). Physicochemical properties evaluated on 1 mg of drug compound were drug content (high-performance liquid chromatography), size [dynamic light scattering (DLS)], morphology (scanning electron microscopy), thermal characteristics (differential scanning calorimetry), and X-ray powder diffraction (XRPD). Scaling down nanosuspension production to 10 mg of drug compound was feasible for the seven model compounds using both designs, the planetary mill design being more robust. Similar results were obtained for both designs upon milling 10 mg of drug compound. Drug content determination was precise and accurate. DLS was the method of choice for size measurements. Morphology evaluation and thermal analysis were feasible, although sample preparation had a big influence on the results. XRPD in capillary mode was successfully performed, both in the suspended state and after freeze-drying in the capillary. Results obtained for the latter were superior. Both the production and the physicochemical evaluation of nanosuspensions can be successfully downscaled, enabling nanosuspension screening applications in preclinical development settings.     

The biofuel potential of municipal solid waste

The world in the 21st century is facing a dual crisis of increasing waste and global climate change. Substituting fossil fuels with waste biomass‐derived cellulosic ethanol is a promising strategy to simultaneously meet part of our energy needs, mitigate greenhouse gas (GHG) emissions, and manage municipal solid waste (MSW). However, the global potential of MSW as an energy source is as yet unquantified. Here, we report increasing trends of MSW generation, and waste biomass‐derived cellulosic ethanol potentials in relation to socio‐economic development across 173 countries, and show that globally, up to 82.9 billion litres of waste paper‐derived cellulosic ethanol can be produced worldwide, replacing 5.36% of gasoline consumption, with accompanying GHG emissions savings of between 29.2% and 86.1%.     

Isolation and Characterization of A Metal-resistant Pseudomonas Aeruginosa Strain

Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin.     

西藏波密柑马蜂的筑巢习性（英文）

柑马蜂Polistes mandarinus在不同的环境中会利用植物材料制作各种不同的蜂巢。我们在2009年5-8月间观察研究了西藏自治区波密县的柑马蜂筑巢习性, 并收集62个蜂巢。柑马蜂利用植物纤维和树脂将它们的巢穴筑于当地居民的木屋檐下。收集的42个在用蜂巢中, 8月末平均拥有79.64±65.28个蜂房, 13.1±10.68个蜂蛹。20个弃用蜂巢中, 平均拥有12.95±3.99个蜂房。8月末, 成功的柑马蜂群拥有约80个个体。研究地的柑马蜂采取3种方式来筑巢, 但以第1种方式最为普遍。第1种筑巢方式筑造的蜂巢以两个蜂房连接巢柄, 结构最为稳定, 所以也应用最广, 后两种筑巢方式筑造的蜂巢均以一个蜂房连接巢柄, 是由于越冬雌蜂在找不到合适的筑巢材料时才采取的建巢方式。     

Effects of substrate composition on the structure of microbial communities in wastewater using fluorescence in situ hybridisation

The microbial composition in a pulp and paper wastewater aerated lagoon system was analysed using fluorescence in situ hybridisation (FISH) to gain further understanding of the effect of substrate composition on microbial diversity for improved management of wastewater treatment systems. Few experiments have been conducted to tease apart the factors influencing the composition and abundance of certain groups within these wastewaters. Specific probes were used to investigate and enumerate the different bacterial groups present at particular stages through the treatment system over an extended period. Community composition and abundance of specific groups differed through the system however temporal stability was retained despite significant variability in the wastewater. Middle stream wastewater samples were enriched to explore the impact of different carbon/nitrogen/phosphorus (C:N:P) ratios on community composition and provide functionality to groups of micro-organisms within the microbial consortia. Nitrogen and phosphorus conditions did not impact community composition of methanol-fed cultures, which exhibited a dominance of Betaproteobacteria (>75%), namely Methylotrophic bacteria. This was confirmed through 16S rRNA gene sequencing and specific FISH probing, reflecting population observations at the beginning of the treatment system. We conclude that the nutrient and carbon combinations used in the enrichments created an interactive effect, altering the community composition and mimicking the main substrate load in the different stages of the treatment system. Finally, pulp and paper wastewater microbial composition was highly variable across the treatment system but was stable within the time sampled, with the enrichments emulating the substrate loads in the full scale system.     

Determination of the lysosomal hydrolase activity in blood collected on filter paper,an alternative to screen high risk populations

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.     

Extended use of a selective inhibitor of acid lipase for the diagnosis of Wolman disease and cholesteryl ester storage disease

Lysosomal acid lipase (LAL) deficiency produces two well defined inborn disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe, early-onset condition involving massive storage of triglycerides and cholesteryl esters in the liver, with death usually occurring before one year of life. CESD is a more attenuated, later-onset disease that leads to a progressive and variable liver dysfunction. Diagnosis of LAL deficiency is mainly based on the enzyme assay of LAL activity in fibroblasts. Recently, a selective acid lipase inhibitor was used for the determination of enzyme activity in dried-blood filter paper (DBFP) samples. To extend and to validate these studies, we tested LAL activity with selective inhibition on DBFP samples, leukocytes and fibroblasts. Our results showed a clear discrimination between patients with LAL deficiency and healthy controls when using DBFP, leukocytes or fibroblasts (p < 0.001). Deficiency of LAL was also demonstrated in individuals referred to our laboratory with suspected clinical diagnosis of WD, CESD, and Niemann–Pick type B. We conclude that the assay of LAL using selective inhibitor is a reliable and useful method for the identification of LAL deficiency, not only in DBFP samples but also in leukocytes and fibroblasts. This is important as enzyme replacement therapy for LAL deficiency is currently being developed, making the correct diagnosis a critical issue.     

互花米草纤维与造纸污泥混合制作污泥纤维板

利用长纤维与造纸污泥混合制作纤维板,以互花米草秸秆、纺织长纤维和废纸浆与造纸污泥混合制浆,经抄纸板机复合热压成型得到工业污泥纤维板材,并分析不同纤维板材的性能差异。结果表明:基于"生物钢筋+水泥"模型,互花米草秸秆纤维与造纸污泥复合成的纤维板材在硬挺度、耐破度、密度和吸水性能上均具有明显优势。此工艺为综合利用造纸污泥等工业废弃污泥提供了可借鉴的工艺技术,并为综合利用秸秆纤维提供了相关参考。