Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway

E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members.     

ProLysED

Bacterial proteases are an important group of enzymes that have very diverse biochemical and cellular functions. Proteases from prokaryotic sources also have a wide range of uses, either in medicine as pathogenic factors or in industry and therapeutics. ProLysED (Prokaryotic Lysis Enzymes Database), our meta-server integrated database of bacterial proteases, is a useful, albeit very niche, resource. The features include protease classification browsing and searching, organism-specific protease browsing, molecular information and visualisation of protease structures from the Protein Data Bank (PDB) as well as predicted protease structures. AVAILABILITY: ProLysED is integrated into the ProLysES (Prokaryotic Lysis Enzymes Site) website at http://genome.ukm.my/prolyses/. Access to the ProLysED database is free for academic users upon registration.     

The value of ischemia-modified albumin compared with d-dimer in the diagnosis of pulmonary embolism

Study objective

The primary aim of this study was to investigate whether IMA levels are helpful in the diagnosis of pulmonary embolism (PE). The secondary aim was to determine whether IMA was more effective alone or in combination with clinical probability scores in the diagnosis of PE. Thirdly, the sensitivity and specificity of IMA is compared with D-dimer both with and without clinical probability scores in patients with suspected PE.

Methods

Consecutive patients presenting to the emergency department with suspected PE were prospectively recruited, and healthy volunteers were also enrolled as controls. D-dimer and IMA levels were measured for the entire study group. Wells and Geneva scores were calculated and s-CTPA was performed on all suspected PE patients.

Results

The study population consisted of 130 patients with suspected PE and 59 healthy controls. Mean IMA levels were 0.362 ± 0.11 ABSU for Group A, the PE group (n = 75); 0.265 ± 0.07 ABSU for Group B, the non-PE group (n = 55); and 0.175 ± 0.05 ABSU for Group C, the healthy control group (p < 0.0001). At a cut-off point of 0.25 ABSU, IMA was 93% sensitive and 75% specific in the diagnosis of PE. PPV was 79.4% and NPV was 78.6%. Mean D-dimer levels were 12.48 ± 10.88 μg/ml for Group A; 5.36 ± 7.80 μg/ml for Group B and 0.36 ± 0.16 μg/ml for Group C (p < 0.0001). The D-dimer cut-off point was 0.81 μg/ml with a sensitivity of 98.9% and a specificity of 62.7%, PPV of 69.4% and NPV of 83.3%. The use of IMA in combination with Wells and Geneva clinical probability scores was determined to have a positive impact on these scores'' sensitivity and negative predictive values.

Conclusion

IMA is a good alternative to D-dimer in PE diagnosis in terms of both cost and efficiency. Used in combination with clinical probability scores, it has a similar positive effect on NPV and sensitivity to that of D-dimer. The PPV of IMA is better than D-dimer, but it is still unable to confirm a diagnosis of PE without additional investigation.     

Biosorption of copper(II) ions onto powdered waste sludge in a completely mixed fed-batch reactor: estimation of design parameters

Biosorption of Cu(II) ions onto pre-treated powdered waste sludge (PWS) was investigated using a fed-batch operated completely mixed reactor. Fed-batch adsorption experiments were performed by varying the feed flow rate ( 0.075-0.325 l h(-1)), feed copper (II) ion concentrations (50-300 mg l(-1)) and the amount of adsorbent (1-6 g PWS) using fed-batch operation. Breakthrough curves describing the variations of effluent copper ion concentrations with time were determined for different operating conditions. Percent copper ion removals from the aqueous phase decreased, but the biosorbed (solid phase) copper ion concentrations increased with increasing the feed flow rate and Cu(II) concentration. A modified Bohart-Adams equation was used to determine the biosorption capacity of PWS and the rate constant for Cu(II) ion biosorption. Adsorption rate constant in fed-batch operation was an order of magnitude larger than those obtained in adsorption columns because of elimination of mass transfer limitations encountered in the column operations while the biosorption capacity of PWS was comparable with powdered activated (PAC) in column operations. Therefore, a completely mixed reactor operated in fed-batch mode was proven to be more advantageous as compared to adsorption columns due to better contact between the phases yielding faster adsorption rates.     

Rapid Clinical Assessment to Facilitate the Triage of Adults with Falciparum Malaria,a Retrospective Analysis

Background

Most adults dying from falciparum malaria will die within 48 hours of their hospitalisation. An essential component of early supportive care is the rapid identification of patients at greatest risk. In resource-poor settings, where most patients with falciparum malaria are managed, decisions regarding patient care must frequently be made using clinical evaluation alone.

Methods

We retrospectively analysed 4 studies of 1801 adults with severe falciparum malaria to determine whether the presence of simple clinical findings might assist patient triage.

Results

If present on admission, shock, oligo-anuria, hypo- or hyperglycaemia, an increased respiratory rate, a decreased Glasgow Coma Score and an absence of fever were independently predictive of death. The variables were used to construct a simple clinical algorithm. When applied to the 1801 patients, this algorithm’s positive predictive value for survival to 48 hours was 99.4 (95% confidence interval (CI) 97.8–99.9) and for survival to discharge 96.9% (95% CI 94.3–98.5). In the 712 patients receiving artesunate, the algorithm’s positive predictive value for survival to 48 hours was 100% (95% CI 97.3–100) and to discharge was 98.5% (95% CI 94.8–99.8).

Conclusions

Simple clinical findings are closely linked to the pathophysiology of severe falciparum malaria in adults. A basic algorithm employing these indices can facilitate the triage of patients in settings where intensive care services are limited. Patients classified as low-risk by this algorithm can be safely managed initially on a general ward whilst awaiting senior clinical review and laboratory data.     

RNase L mediates transient control of the interferon response through modulation of the double-stranded RNA-dependent protein kinase PKR

The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2alpha, eIF2alpha, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2alpha appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses.     

Stereospecific synthesis of a new class of compounds: bis-homoconduritol-A, -D, and -F

Bis-homoconduritol derivatives with conduritol-A, -D, and -F structures have been synthesized starting from cyclooctatetraene. The photooxygenation of trans-7,8-dibromo- and cis-7,8-dichlorobicyclo[4.2.0]octa-2,4-dienes afforded the bicyclic endoperoxides. Reduction of the endoperoxides with thiourea followed by acetylation gave the corresponding diacetates. The KMnO(4) oxidation and epoxidation of the diacetates followed by acetylation gave the tetraacetates. Removal of the halides either with zinc-dust or Na-anthracene followed by the ammonolysis of tetraacetates afforded the bis-homoconduritol derivatives in high yield.     

Electrophysiological response of chicken’s jejunal epithelium to increasing levels of T-2 toxin

The present investigations were conducted to test the effects of T-2 toxin on electrophysiological variables of jejunal epithelium of chicken. Jejunal segments of broilers were monitored in Ussing chambers in the presence of T-2 toxin at the levels of 0 (negative control), 0 (methanol/vehicle control), 0.1, 1, 5, and 10 μg/ml of buffer. T-2 toxin did not affect basal values of short circuit current (I_sc), transmural potential difference, or tissue conductivity in the jejunal epithelium. T-2 toxin also did not statistically affect glucose-induced electrophysiological variables during the first 3 min of glucose induction. Compared to the vehicle control, the ouabain-sensitive I_sc was negatively affected (P?=?0.008) only under 5 μg of T-2 toxin/ml. Increasing levels of T-2 toxin negatively affected the ouabain-sensitive I_sc in a cubic (P?=?0.007) fashion. These data indicate that acute exposure to moderate levels of T-2 toxin may progressively impair the cation gradient across the jejunal epithelium.     

Genome-Wide Association Studies and Heritability Estimates of Body Mass Index Related Phenotypes in Bangladeshi Adults

Many health outcomes are influenced by a person''s body mass index, as well as by the trajectory of body mass index through a lifetime. Although previous research has established that body mass index related traits are influenced by genetics, the relationship between these traits and genetics has not been well characterized in people of South Asian ancestry. To begin to characterize this relationship, we analyzed the association between common genetic variation and five phenotypes related to body mass index in a population-based sample of 5,354 Bangladeshi adults. We discovered a significant association between SNV rs347313 (intron of NOS1AP) and change in body mass index in women over two years. In a linear mixed-model, the G allele was associated with an increase of 0.25 kg/m² in body mass index over two years (p-value of 2.3·10⁻⁸). We also estimated the heritability of these phenotypes from our genotype data. We found significant estimates of heritability for all of the body mass index-related phenotypes. Our study evaluated the genetic determinants of body mass index related phenotypes for the first time in South Asians. The results suggest that these phenotypes are heritable and some of this heritability is driven by variation that differs from those previously reported. We also provide evidence that the genetic etiology of body mass index related traits may differ by ancestry, sex, and environment, and consequently that these factors should be considered when assessing the genetic determinants of the risk of body mass index-related disease.     

Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation,gene and protein expression

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.