Identification and Validation of Urinary Biomarkers for Differential Diagnosis and Evaluation of Therapeutic Intervention in Anti-neutrophil Cytoplasmic Antibody-associated Vasculitis

Renal activity and smoldering disease is difficult to assess in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) because of renal scarring. Even repeated biopsies suffer from sampling errors in this focal disease especially in patients with chronic renal insufficiency. We applied capillary electrophoresis coupled to mass spectrometry toward urine samples from patients with active renal AAV to identify and validate urinary biomarkers that enable differential diagnosis of disease and assessment of disease activity. The data were compared with healthy individuals, patients with other renal and non-renal diseases, and patients with AAV in remission. 113 potential biomarkers were identified that differed significantly between active renal AAV and healthy individuals and patients with other chronic renal diseases. Of these, 58 could be sequenced. Sensitivity and specificity of models based on 18 sequenced biomarkers were validated using blinded urine samples of 40 patients with different renal diseases. Discrimination of AAV from other renal diseases in blinded samples was possible with 90% sensitivity and 86.7–90% specificity depending on the model. 10 patients with active AAV were followed for 6 months after initiation of treatment. Immunosuppressive therapy led to a change of the proteome toward “remission.” 47 biomarkers could be sequenced that underwent significant changes during therapy together with regression of clinical symptoms, normalization of C-reactive protein, and improvement of renal function. Proteomics analysis with capillary electrophoresis-MS represents a promising tool for fast identification of patients with active AAV, indication of renal relapses, and monitoring for ongoing active renal disease and remission without renal biopsy.Systemic vasculitides are a heterogeneous group of disorders with inflammation of the blood vessel wall as their common hallmark. These disorders often pose difficulties with regard to diagnosis and monitoring of disease activity both at the initial presentation and during follow-up. In one subgroup of small vessel vasculitides, the advent of anti-neutrophil cytoplasmic antibodies (ANCAs)¹ in the 1980s not only provided a new pathogenetic concept but also a diagnostic marker (1, 2). In this group, the granulomatous ANCA-associated vasculitis (GAAV) (previously named Wegener granulomatosis) and the microscopic polyangiitis (MPA) share several common features, including pauci-immune focal crescentic necrotizing glomerulonephritis and often a pulmonary capillaritis (3). Because of the association with ANCA, these diseases (together with the Churg-Strauss syndrome) are sometimes collectively referred to as ANCA-associated vasculitis (AAV). Recently circulating endothelial cells have emerged as an important marker correlating with severity and activity of the systemic vasculitic disease, and their clinical use in small vessel AAV has been demonstrated (4, 5). Regarding renal involvement, which is found in up to 80–90% of the patients with AAV, activity is defined by kidney biopsy with pauci-immune necrotizing glomerulonephritis. Renal involvement may occur or recur at every point of the disease and the follow-up even if other organ involvement is controlled by immunosuppressive therapy. Early detection is important as renal prognosis depends on early administration of immunosuppressive treatment (6), and scarring and relapses increase the risk for terminal renal failure, which itself is a risk factor for patient survival. As kidney biopsy is invasive and the risk of bleeding increases with chronic renal damage, surrogate markers, such as rising creatinine, increasing proteinuria, and most importantly erythrocytes and erythrocyte cast in the urinary sediment, are used. However, these markers have limitations. Microhematuria might persist despite remission, proteinuria might increase despite improvement in renal function, and other renal diseases can also develop (7). Therefore, new markers for renal disease activity are eagerly awaited.Recently proteome analysis of urine has presented itself as a promising tool in the definition of chronic renal diseases (8, 9). We have developed an analytical platform for human urine analysis using capillary electrophoresis (CE) coupled on line to an ESI-TOF mass spectrometer (8, 10, 11). This approach permits the rapid analysis of the low molecular weight urinary proteome/peptidome in a single step and has enabled identification and validation of several urinary biomarkers in patients with different renal diseases (11–14).In this study, we aimed toward identification of biomarkers for active AAV and response to immunosuppressive treatment. The results indicate that urinary proteome analysis enables differential diagnosis and monitoring of renal disease activity of AAV.     

Genome analysis technologies: towards species identification by genotype.

Traditional identification of species has been based on phenotypic traits, although it is clear that, theoretically, genotype-based classification is more accurate. This is especially the case for microorganisms which possess less identifiable traits and are more easily influenced by environment. Therefore, technology that allows identification of species based on genotype is highly desirable. Whole genome sequencing can provide a sufficient amount of information and can be determinative for this purpose but is very impractical for routine use. Thus, a competent technology is needed that allows a reproducible reduction in the amount of information required about a whole genome, while still providing sufficiently accurate identification. It is almost imperative for such a technology to be of a high cost-performance and of easy handling. Universality and portability are also strongly desired. Based on these criteria, the current state of genome analysis technologies are reviewed. Among various methodologies discussed here, amplified fragment length polymorphism (AFLP), genome profiling (GP) and microarrays are the subject of particular attention. As species identification is a base for most fields of biology including microbiology, ecology, epidemiology and for various biotechnologies, it is of paramount importance to establish a more efficient, easily handled and more objective methodology, in parallel with conventional phenotype-based methodologies. GP is currently considered to have the most optimal nature for identification of species since it can reproducibly reduce a huge amount of genome information to a manageable size by way of random polymerase chain reaction and can extract a sufficient amount of information for species identification from the DNA fragments thus profiled by temperature gradient gel electrophoresis. The potential ability of DNA microarrays for this purpose is also discussed and promises much for the future.     

Mechanism of the CO-sensing heme protein CooA: new insights from the truncated heme domain and UVRR spectroscopy

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.     

Effect of gut bacterial isolates from Apis mellifera jemenitica on Paenibacillus larvae infected bee larvae

The probiotic effects of seven newly isolated gut bacteria, from the indegenous honey bees of Saudi Arabia were investigated. In vivo bioassays were used to investigate the effects of each gut bacterium namely, Fructobacillus fructosus (T1), Proteus mirabilis (T2), Bacillus licheniformis (T3), Lactobacillus kunkeei (T4), Bacillus subtilis (T5), Enterobacter kobei (T6), and Morganella morganii (T7) on mortality percentage of honey bee larvae infected with P. larvae spores along with negative control (normal diet) and positive control (normal diet spiked with P. larvae spores). Addition of gut bacteria to the normal diet significantly reduced the mortality percentage of the treated groups. Mortality percentage in all treated groups ranged from 56.67% up to 86.67%. T6 treated group exhibited the highest mortality (86.67%), whereas T4 group showed the lowest mortality (56.67%). Among the seven gut bacterial treatments, T4 and T3 decreased the mortality 56.67% and 66.67%, respectively, whereas, for T2, T6, and T7 the mortality percentage was equal to that of the positive control (86.67%). Mortality percentages in infected larval groups treated with T1, and T5 were 78.33% and 73.33% respectively. Most of the mortality occurred in the treated larvae during days 2 and 3. Treatments T3 and T4 treatments showed positive effects and reduced mortality.     

Assessment of the contribution of climate change and human activities to desertification in Northern Kordofan-Province,Sudan using net primary productivity as an indicator

Using net primary productivity (NPP) as an indicator to desertification driving factor and expansions is one of the importance tools in the assessment of the contribution of climate change and human activity in desertification. In this study we used three types of net primary productivity; the actual NPP, Potential NPP and HNPP (human appropriation of NPP) to discriminate the relative role of climate change and human activities in desertification from 2000 to 2008 in Northern Kordofan province-Sudan. The results showed, 63.75% of the study area experienced desertification expansion. Within which, 67.32% was induced by Human activities compared with 32.03% caused by climate change and 0.65% caused by a combination of the two factors. By contrast, climate change is the dominant factor of desertification reversion, 2.3% of desertification reversion caused by human activities compared with 97.7% induced by climate change and there isn’t interaction between climate change and human activities in reversion area. The largest area of expansion and reversion occurred in northeast and western parts of the study area respectively. We developed two propositions in the study area. First, the desertification expansion was induced by human activities, whereas desertification reversion was induced by climate change as typified in north south part, central part and western part. Second, both desertification expansion and reversion was induced by climate change as typified in northeast part of study area.     

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.     

Peracetylated 1,6-dibromo-D-glucitol as efficient precursor of 1,6-diiodo and some mono-, disubstituted and heterocyclic D-glucitol derivatives

2,3,4,5-Tetra-O-acetyl-1,6-dibromo-1,6-dideoxy-D-glucitol (1a) obtained from D-glucitol was easily transformed into the 1,6-diiodo derivative in excellent yield (97%) by reaction with an excess of sodium iodide in refluxing butanone in 2 h. When the reaction time was prolonged to 24 h and the crude product was acetylated, 1,2,3,4,5-penta-O-acetyl-6-deoxy-6-iodo-D-glucitol and D-glucitol hexaacetate were isolated in 50 and 26% yields, respectively. The monodehalogenation then took place regioselectively at C-1. This regioselectivity allowed the synthesis of some mono- and disubstituted derivatives of D-glucitol. Thus, the peracetylated derivatives of D-glucitol, 6-bromo, 6-bromo-1-S-butyl, 6-bromo-1-S-octyl, 6-S-butyl, 6-S-butyl-1-S-octyl, 1-S-butyl, 1,6-di-S-octyl and 6-S-phenyl were synthesised in good to excellent yields. With S= as binucleophilic reagent, 1a gave mainly the thiepane derivative (75%) plus the 1-S-acetyl-2,6-anhydro-D-glucitol derivative as a by-product (10%).     

Phosphorus deficiency increases nodule phytase activity of faba bean–rhizobia symbiosis

The effect of phosphorus deficiency on growth, nodulation and phytase activity was studied in glasshouse for four symbioses involving two faba bean cultivars, namely Aguadulce (AG) and Alfia (AL), and two local rhizobial isolates, namely RhF1 and RhF2. The P deficiency was applied by adding 25 µmol of Pi plant^?1 week^?1 to nutrient solution, whereas the sufficient control received 125 µmol plant^?1 week^?1. At flowering stage, the plants were harvested for assessment of growth and nodulation, P and N contents in organs as well as activities of phytase and phosphatases in nodules. The latter were highly stimulated by P deficiency, particularly for AL–RhF1 symbiosis for which shoot growth and P content were not affected by P deficiency. Using in situ RT-PCR, the expression of a plant histidine acid phytase HAP gene was detected in the nodule cortex under P deficiency. It is concluded that high nodule phytase activity constitutes a mechanism for faba bean plants to adapt their nitrogen fixation to P deficiency.