In situ proteolysis for protein crystallization and structure determination.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Use of plasma DNA to predict mortality and need for intensive care in patients with abdominal pain.

BACKGROUND: We investigated the value of plasma deoxyribonucleic acid concentrations in patients presenting with acute abdominal pain to predict need for intensive care or mortality. METHODS: Plasma deoxyribonucleic acid taken from patients with acute abdominal pain was analyzed for the beta-globin gene using the quantitative polymerase chain reaction. The primary outcome measure was the combined 28-day mortality or admission to the intensive care unit. RESULTS: Of 287 consecutive patients with acute abdominal pain recruited, 12 patients were admitted to the intensive care unit and/or died. Median plasma DNA concentrations were higher in patients with cancer and major organ inflammation. Mean plasma DNA concentrations were three-fold higher in patients with systemic inflammatory response syndrome, five-fold higher in patients who died within 28 days, and eight-fold higher in patients admitted to the intensive care unit. The area under the receiver operator curve for plasma DNA concentrations and intensive care unit admission/mortality was 0.804. At a cut-off of 1100 GE/ml, the sensitivity was 67% (95%CI 35-90) and specificity was 89% (95%CI 84-92). At a cut-off of 175 GE/ml, the sensitivity was 100% (95%CI 73-100) and specificity was 30% (95%CI 25-36). Plasma DNA concentration predicted need for intensive care unit admission or death (adjusted odds ratio 1.4; P<0.0001). CONCLUSIONS: Plasma DNA may have a role in patients with acute abdominal pain as a marker for inflammation and cancer, and a predictor of intensive care unit admission/mortality.

Crystal structure of CTP:glycerol-3-phosphate cytidylyltransferase from Staphylococcus aureus: examination of structural basis for kinetic mechanism.

Integrity of the cell wall is essential for bacterial survival, and as a consequence components involved in its biosynthesis can potentially be exploited as targets for antibiotics. One such potential target is CTP:glycerol-3-phosphate cytidylyltransferase. This enzyme (TarD(Sa) in Staphylococcus aureus and TagD(Bs) in Bacillus subtilis) catalyzes the formation of CDP-glycerol, which is used for the assembly of linkages between peptidoglycan and teichoic acid polymer in Gram-positive bacteria. Intriguingly, despite the high sequence identity between TarD(Sa) and TagD(Bs) (69% identity), kinetic studies show that these two enzymes differ markedly in their kinetic mechanism and activity. To examine the basis for the disparate enzymological properties, we have determined the crystal structure of TarD(Sa) in the apo state to 3 A resolution, and performed equilibrium sedimentation analysis. Comparison of the structure with that of CTP- and CDP-glycerol-bound TagD(Bs) crystal structures reveals that the overall structure of TarD(Sa) is essentially the same as that of TagD(Bs), except in the C-terminus, where it forms a helix in TagD(Bs) but is disordered in the apo TarD(Sa) structure. In addition, TarD(Sa) can exist both as a tetramer and as a dimer, unlike TagD(Bs), which is a dimer. These observations shed light on the structural basis for the differing kinetic characteristics between TarD(Sa) and TagD(Bs).

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families.

Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.

Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi.

Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD(+)-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP(+) as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.

A comparison of emergency department utilization by elderly and younger adult patients presenting to three hospitals in Hong Kong.

BACKGROUND: The elderly population is increasing in absolute and relative terms in most developed countries, and this is protected to have a major impact on the delivery of health care, particularly acute and emergency services. The aim of this study is to describe the pattern of utilization of emergency department (ED) services in Hong Kong by the elderly and to compare it to the utilization by younger adults. METHODS: Data on ED visits to three acute hospitals in the eastern New Territories were retrieved from a central computerized database of ED attendances. Data on all adult patients (aged > or =15 years) who attended the three EDs in 2006 were analyzed retrospectively. Patients aged 15 to 64 years were defined as younger adults; patients aged > or =65 years were defined as elderly. The attendance rate, ED consultation process, hospital admission rate and disease pattern of the two age groups were compared. RESULTS: Elderly patients required significantly more emergency care resources than younger adults. Elderly ED patients were brought to hospital more frequently by ambulance (42.8% vs. 14.8%, p < 0.0001) and required hospital admission more often (45.0% vs. 15.5%, p < 0.0001) than younger adults. A significantly higher proportion of elderly patients were triaged as being in the critical, emergency or urgent categories compared to younger adults (44.4% vs. 18.2%, p < 0.0001). Laboratory tests, radiography and CT scanning were performed on elderly patients more frequently than on younger adults (p < 0.0001), and their lengths of stay in EDs and emergency wards were significantly longer (p < 0.0001). Neurological symptoms and chest pain were the most common presenting symptoms in elderly ED patients. CONCLUSION: With the foreseeable rapid growth of the elderly population, ED utilization by the elderly will increase. Health service delivery, including that in the ED, needs to take account of the specific features and requirements of the elderly population in each locale.

Five year analysis of Jockey Club horse-related injuries presenting to a trauma centre in Hong Kong.

BACKGROUND: Professional horse riding-related injuries have not been studied before in Hong Kong, although horse racing takes place very regularly in the territory. In addition, the equestrian events of the 2008 Beijing Olympic Games will come to Hong Kong. This study analysed the pattern of horse-related injury among patients who presented to a trauma centre in a teaching hospital in Hong Kong. METHODS: Information from the trauma centre database was analysed retrospectively. The database includes trauma patients who had sustained potentially severe injuries that warranted initial assessment and resuscitation in a trauma resuscitation room (triage category 1 or 2). Data analysed included demographic variables, causes and mechanisms of injury, anatomical injuries, anatomical and physiological trauma scores, and patient outcome. RESULTS: Between January 2001 and June 2005, 2312 trauma patients were entered into the database. Thirty-six (1.6%) patients had sustained horse-related injuries (mean age 34 years, range 17-54; male to female ratio 32:4), all whilst at work in the Jockey Club. Twenty-two patients were injured between midnight and 09:00 h. This group stayed in the resuscitation room for longer prior to admission compared with patients presenting between 09:00h and midnight (median time 127 min (interquartile range [IQR] 57-183) versus 58 min (IQR 43-83), p=0.06). Twenty-five patients fell from horseback, whilst 11 were kicked by the horse. Twenty patients had a single injury and 16 patients had multiple injuries. Eighteen patients had injuries to the thorax, abdomen, thoracolumbar spine or pelvis. Eleven patients had head, face and cervical spine injuries and 11 had limb injuries. Twenty-five patients were admitted, including four admitted to the intensive care unit (ICU). Ten patients required surgery. Median (standard deviation [S.D.]) probability of survival was 0.996 (0.052) and median revised trauma score (RTS) (S.D.) was 7.841 (0.624). There were no fatalities. Potentially serious horse-related injuries presented once every 6 weeks. CONCLUSION: Most injuries are minor and affect the trunk but occur out of hours. Helmets, face shields and body protectors should be worn when riding or handling horses.