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2.  Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome▿† 
Applied and Environmental Microbiology  2011;77(21):7595-7604.
The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
doi:10.1128/AEM.05495-11
PMCID: PMC3209177  PMID: 21908633
5.  Survival of HIV‐infected patients in the intensive care unit in the era of highly active antiretroviral therapy 
Thorax  2007;62(11):964-968.
Background
Several studies have described improved outcomes for HIV‐infected patients admitted to the intensive care unit (ICU) since the introduction of highly active antiretroviral therapy (HAART). A study was undertaken to examine the outcome from the ICU for HIV‐infected patients and to identify prognostic factors.
Methods
A retrospective study of HIV‐infected adults admitted to a university affiliated hospital ICU between January 1999 and December 2005 was performed. Information was collected on patient demographics, receipt of HAART (no patient began HAART on the ICU), reason for ICU admission and hospital course. Outcomes were survival to ICU discharge and to hospital discharge.
Results
102 patients had 113 admissions to the ICU; HIV infection was newly diagnosed in 31 patients. Survival (first episode ICU discharge and hospital discharge) was 77% and 68%, respectively, compared with 74% and 65% for general medical patients. ICU and hospital survival was 78% and 67% in those receiving HAART, and 75% and 66% in those who were not. In univariate analysis, factors associated with survival were: haemoglobin (OR = 1.25, 95% CI 1.03 to 1.51, for an increase of 1 g/dl), CD4 count (OR = 1.59, 95% CI 0.98 to 2.58, for a 10‐fold increase in cells/µl), APACHE II score (OR = 0.51, 95% CI 0.29 to 0.90, for a 10 unit increase) and mechanical ventilation (OR = 0.29, 95% CI 0.10 to 0.83).
Conclusions
The outcome for HIV‐infected patients admitted to the ICU was good and was comparable to that in general medical patients. More than a quarter of patients had newly diagnosed HIV infection. Patients receiving HAART did not have a better outcome.
doi:10.1136/thx.2006.072256
PMCID: PMC2117109  PMID: 17517829
6.  Pharmacological optimization of tissue perfusion 
After fluid resuscitation, vasoactive drug treatment represents the major cornerstone for correcting any major impairment of the circulation. However, debate still rages as to the choice of agent, dose, timing, targets, and monitoring modalities that should optimally be used to benefit the patient yet, at the same time, minimize harm. This review highlights these areas and some new pharmacological agents that broaden our therapeutic options.
doi:10.1093/bja/aep135
PMCID: PMC2700012  PMID: 19460775
arterial pressure, drug effects; cardiovascular system, effects; heart, cardiac output; oxygen, therapy; pharmacology, agonists adrenergic
8.  Improved survival for HIV infected patients with severe Pneumocystis jirovecii pneumonia is independent of highly active antiretroviral therapy 
Thorax  2006;61(8):716-721.
Background
Despite a decline in incidence of Pneumocystis jirovecii pneumonia (PCP), severe PCP continues to be a common cause of admission to the intensive care unit (ICU) where mortality remains high. A study was undertaken to examine the outcome from intensive care for patients with PCP and to identify prognostic factors.
Methods
A retrospective cohort study was conducted of HIV infected adults admitted to a university affiliated hospital ICU between November 1990 and October 2005. Case note review collected information on demographic variables, use of prophylaxis and highly active antiretroviral therapy (HAART), and hospital course. The main outcome was 1 month mortality, either on the ICU or in hospital.
Results
Fifty nine patients were admitted to the ICU on 60 occasions. Thirty four patients (57%) required mechanical ventilation. Overall mortality was 53%. No patient received HAART before or during ICU admission. Multivariate analysis showed that the factors associated with mortality were the year of diagnosis (before mid 1996 (mortality 71%) compared with later (mortality 34%; p = 0.008)), age (p = 0.016), and the need for mechanical ventilation and/or development of pneumothorax (p = 0.031). Mortality was not associated with sex, ethnicity, prior receipt of sulpha prophylaxis, haemoglobin, serum albumin, CD4 count, Pao2, A‐ao2 gradient, co‐pathology in bronchoscopic lavage fluid, medical co‐morbidity, APACHE II score, or duration of mechanical ventilation.
Conclusions
Observed improved outcomes from severe PCP for patients admitted to the ICU occurred in the absence of intervention with HAART and probably reflect general improvements in ICU management of respiratory failure and ARDS rather than improvements in the management of PCP.
doi:10.1136/thx.2005.055905
PMCID: PMC2104703  PMID: 16601092
AIDS; intensive care; mechanical ventilation;  Pneumocystis jirovecii ; opportunistic infections; respiratory failure
9.  Gene expression of interleukin 18 in unstimulated peripheral blood mononuclear cells of patients with alcoholic cirrhosis 
Gut  2001;49(1):106-111.
BACKGROUND—Most patients with alcohol induced cirrhosis (AC) and chronic endotoxinaemia are not suffering from clinically evident systemic inflammatory reactions. This may be due to altered gene expression of cytokines, possibly related to endotoxin (for example, tolerance and sensitisation). Interleukin 18 (IL-18; interleukin γ inducing factor) modulates local cytokine production in response to endotoxin (lipopolysaccharide (LPS)).
AIM—To investigate the systemic immune response of patients with AC and to see if unstimulated peripheral blood mononuclear cells (PBMC) from patients with AC are activated and contribute to gene expression of IL-18.
METHODS—Plasma levels of endotoxin (LPS) and serum levels of IL-18 were measured by enzyme linked immunoassay and the amoebocyte lysate test in 74 abstinent patients with different stages of AC (Child-Pugh stage A, n=18; B, n=22; C, n=34) and compared with healthy controls (n=43). Gene expression of IL-18 was assessed by semiquantitative reverse transcription-polymerase chain reaction in freshly isolated unstimulated PBMC of a subgroup of 14 patients with AC compared with five healthy controls.
RESULTS—Gene expression of IL-18 specific mRNA in unstimulated PBMC was significantly enhanced in patients with advanced AC (Child-Pugh stage C) and correlated with plasma LPS and serum CD14 levels (Spearman rank correlation factors r=0.76 and r=0.72). Serum concentrations of IL-18 were also elevated compared with healthy controls (p<0.001) but correlation with serum levels of CD14 and plasma levels of LPS was much weaker compared with mRNA data (Spearman rank correlation factors r=0.47 and r=0.26).
CONCLUSIONS—Our in vivo data suggest a presensitisation of "unstimulated" PBMC in the circulation of patients with AC by endotoxin. The term "unstimulated" may be inadequate in patients with AC. Further investigations are needed to define the exact mechanisms and localisation of sensitisation of PBMC in vivo.


Keywords: liver cirrhosis; peripheral blood mononuclear cells; interleukin 18; lipopolysaccharide
doi:10.1136/gut.49.1.106
PMCID: PMC1728343  PMID: 11413118
10.  Cardiac output in 1998 
Heart  1998;79(5):425-428.
PMCID: PMC1728678  PMID: 9659184
13.  Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides. 
Nucleic Acids Research  1999;27(24):e38.
Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.
PMCID: PMC148761  PMID: 10572190
14.  Olfactory Receptor Database: a database of the largest eukaryotic gene family. 
Nucleic Acids Research  1999;27(1):343-345.
The Olfactory Receptor Database (ORDB) is a WWW-accessible database that stores data on Olfactory Receptor-like molecules (ORs) and has been open to the public since June 1996. It contains a public and a private area. The public area includes published DNA and protein sequence data for ORs, links to OR models and data on their expression, chromosomal localization and source organism, as well as (i) links to bibliography through PubMed and (ii) interactive WWW-based tools, such as BLAST homology searching. The private area functions as a service to laboratories that are actively cloning receptors. Source laboratories enter the sequences of the receptor clones they have characterized to the private database and can search for identical or near identical OR sequences in both public and private databases. If another laboratory has cloned and deposited an identical or closely matching sequence there are means for communication between the laboratories to help avoid duplication of work. ORDB is available via the WWW at http://crepe.med.yale.edu/ORDB/HTML
PMCID: PMC148178  PMID: 9847223
15.  The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain. 
Nucleic Acids Research  1998;26(15):3528-3535.
Heterologous expression of human LINE-1 ORF2 in yeast yielded a single polypeptide (Mr145 000) which reacted with specific antibodies and co-purified with a reverse transcriptase activity not present in the host cells. Various deletion derivatives of the ORF2 polypeptide were also synthesized. Reverse transcriptase assays using synthetic polynucleotides as template and primer revealed that ORF2 protein missing a significant portion of the N-terminal endonuclease domain still retains some activity. Deletion of the C-terminal cysteine-rich motif reduces activity only a small amount. Three non-overlapping deletions spanning 144 amino acids just N-terminal to the common polymerase domain of the ORF2 protein were analyzed for their effect on reverse transcriptase activity; this region contains the previously-noted conserved Z motif. The two deletions most proximal to the polymerase domain eliminate activity while the third, most-distal deletion had no effect. An inactive enzyme was also produced by substitution of two different amino acids in a highly-conserved octapeptide sequence, Z8, located within the region removed to make the deletion most proximal to the polymerase domain; substitution of a third had no effect. We conclude that the octapeptide sequence and neighboring amino acids in the Z region are essential for reverse transcriptase activity, while the endonuclease and cysteine-rich domains are not absolutely required.
PMCID: PMC147723  PMID: 9671814
16.  Pharmacy access to syringes among injecting drug users: follow-up findings from Hartford, Connecticut. 
Public Health Reports  1998;113(Suppl 1):81-89.
OBJECTIVE: To break the link between drug use and the human immunodeficiency virus (HIV), in 1992 the state of Connecticut rescinded a 14-year ban on pharmacy sales of syringes without a physician's prescription. In 1993, the Center for Disease Control and Prevention (CDC) evaluated the impact of the new legislation on access to syringes among injecting drug users (IDUs) and found an initial pattern of expanded access. However, it also found that some pharmacies, after negative experiences with IDU customers, reverted to requiring a prescription. This chapter reports findings from a four-year follow-up study of current IDU access to over-the-counter (OTC) pharmacy syringes in Hartford, Connecticut. METHODS: Through structured interviews, brief telephone interviews, and mailed surveys, data on nonprescription syringe sale practices were collected on 27 pharmacies, including 18 of the 21 pharmacies in Hartford and none from pharmacies in contiguous towns, during June and July 1997. Interview data on pharmacy syringe purchase from two sample of IDUs, a group of out-of-treatment injectors recruited through street outreach, and a sample of users of the Hartford Needle Exchange Program, also are reported. RESULTS: The study found that, while market trends as well as negative experiences have further limited pharmacy availability of nonprescription syringes, pharmacies remain an important source of sterile syringes for IDUs. However, the distribution of access in not even; in some areas of the city it is much easier to purchase nonprescription syringes than in other. All of the seven pharmacies located on the north end of Hartford reported that they had a policy of selling OTC syringes, whereas only six (54.5%) of the II pharmacies located on the south end have such a policy. Overt racial discrimination was not found to be a barrier to OTC access to syringes. CONCLUSIONS: To further decrease acquired immunodeficiency syndrome (AIDS) risk among IDUs, there is a need for public education to counter empirically unsupported stereotypes about IDUs that diminish their access to health care and AIDS prevention resources and services. In states or cities where pharmacy sale of nonprescription syringes is illegal, policy makers should examine the benefits of removing existing barriers to sterile syringe acquisition. In cases in which pharmacy sale of nonprescription syringes is legal, local health departments should implement educational programs to inform pharmacy staff and management about the critically important role low-cost (or cost-free), sterile syringe access can play in HIV prevention.
PMCID: PMC1307730  PMID: 9722813
17.  High dependency units in the UK: variable size, variable character, few in number. 
Postgraduate Medical Journal  1995;71(834):217-221.
An exploratory descriptive survey was conducted to determine the size and character of high dependency units (HDUs) in the UK. A telephone survey and subsequent postal questionnaire was sent to the 39 general HDUs in the UK determined by a recent survey from the Royal College of Anaesthetists; replies were received from 28. Most HDUs (82%, n = 23) were geographically distinct from the intensive care unit and varied in size from three to 13 beds, although only 64% (n = 18) reported that all beds were currently open. Nurse: patient ratios were at least 1:3. Fifty per cent of units had one or more designated consultants in charge, although only 11% (n = 3) had specifically designated consultant sessions. Junior medical cover was provided mainly by the on-call speciality term. Twenty units acted as a step-down facility for discharged intensive care unit patients and 21 offered a step-up facility for patients from general wards. Provision of facilities and levels of monitoring varied between these units. Few HDUs exist in the UK and they are variable in size and in the facilities and monitoring procedures which they provide. Future studies are urgently required to determine cost-effectiveness and outcome benefit of this intermediate care facility.
Images
PMCID: PMC2398078  PMID: 7784281
18.  Immunotherapy in the management of sepsis. 
Postgraduate Medical Journal  1995;71(832):71-78.
The pathophysiological effects of severe sepsis, septic shock and related syndromes result from tissues damaged by the uncontrolled production of the mediators of inflammation. Early deaths are related primarily to the acute effects of the systemic inflammatory response. Later deaths are related more closely to the consequences of multiple organ dysfunction. Monoclonal antibodies and other immunotherapies have been developed against bacterial products, cytokines and other mediators involved in this systemic inflammatory response. Immunotherapies may improve outcome in the critically ill with sepsis if used early and as part of the therapeutic regimen of antimicrobial agents and intensive care support.
PMCID: PMC2397941  PMID: 7724438
19.  Intraoperative intravascular volume optimisation and length of hospital stay after repair of proximal femoral fracture: randomised controlled trial. 
BMJ : British Medical Journal  1997;315(7113):909-912.
OBJECTIVES: To assess whether intraoperative intravascular volume optimisation improves outcome and shortens hospital stay after repair of proximal femoral fracture. DESIGN: Prospective, randomised controlled trial comparing conventional intraoperative fluid management with repeated colloid fluid challenges monitored by oesophageal Doppler ultrasonography to maintain maximal stroke volume throughout the operative period. SETTING: Teaching hospital, London. SUBJECTS: 40 patients undergoing repair of proximal femoral fracture under general anaesthesia. INTERVENTIONS: Patients were randomly assigned to receive either conventional intraoperative fluid management (control patients) or additional repeated colloid fluid challenges with oesophageal Doppler ultrasonography used to maintain maximal stroke volume throughout the operative period (protocol patients). MAIN OUTCOME MEASURES: Time declared medically fit for hospital discharge, duration of hospital stay (in acute bed; in acute plus long stay bed), mortality, perioperative haemodynamic changes. RESULTS: Intraoperative intravascular fluid loading produced significantly greater changes in stroke volume (median 15 ml (95% confidence interval 10 to 21 ml)) and cardiac output (1.2 l/min (0.1 to 2.3 l/min)) than in the conventionally managed group (-5 ml (-10 to 1 ml) and -0.4 l/min (-1.0 to 0.2 l/min)) (P < 0.001 and P < 0.05, respectively). One protocol patient and two control patients died in hospital. In the survivors, postoperative recovery was significantly faster in the protocol patients, with shorter times to being declared medically fit for discharge (median 10 (9 to 15) days v 15 (11 to 40) days, P < 0.05) and a 39% reduction in hospital stay (12 (8 to 13) days v 20 (10 to 61) days, P < 0.05). CONCLUSIONS: Proximal femoral fracture repair constitutes surgery in a high risk population. Intraoperative intravascular volume loading to optimal stroke volume resulted in a more rapid postoperative recovery and a significantly reduced hospital stay.
PMCID: PMC2127619  PMID: 9361539
20.  Interleukin-12 induction of Th1 cytokines is important for viral clearance in chronic hepatitis B. 
Journal of Clinical Investigation  1997;99(12):3025-3033.
Interleukin-12, a cytokine with an important role against intracellular pathogens, promotes Th1 cell development, cellmediated cytotoxicity, and interferon-gamma production. We investigated the immunoregulatory role of IL-12 in 72 chronic hepatitis B virus (HBV) carriers, 33 of whom were monitored longitudinally during interferon-alpha treatment. Serum levels of IL-12 heterodimer, IL-12 p40 subunit, IL-4, and Th1 cytokines were determined by specific ELISAs, and hepatitis B core antigen-specific T cell response by a proliferation assay. Chronic HBV carriers had higher serum levels of IL-12 and IL-12 p40 in comparison with controls (P < 0.01), suggesting that IL-12 production is not impaired. The longitudinal analysis revealed a further substantial increase (> 2.5x baseline level) of bioactive IL-12 and Th1 cytokines in patients who cleared HBV and seroconverted to anti- hepatitis B e, unlike the 23 nonresponders with persistent HBV replication (P < 0.01). The IL-12 peak followed the peak of hepatocytolysis by 9.8+/-2.8 wk and occurred either before or simultaneously with hepatitis B e seroconversion. Hepatitis B core antigen-specific T cell proliferation closely correlated with hepatocytolysis and increased significantly in all patients (8 responders and 15 nonresponders) who developed hepatitis flare, irrespective of the virological outcome. These results provide in vivo evidence that IL-12 may have an important role for viral clearance in chronic HBV infection.
PMCID: PMC508155  PMID: 9185527
21.  Alcoholic beverages produced by alcoholic fermentation but not by distillation are powerful stimulants of gastric acid secretion in humans. 
Gut  1997;40(1):49-56.
BACKGROUND: The effect of commonly ingested alcoholic beverages on gastric acid output and release of gastrin in humans is unknown. AIM AND METHODS: In 16 healthy humans the effect of some commonly ingested alcoholic beverages produced by fermentation plus distillation (for example, whisky, cognac, calvados, armagnac, and rum) or by alcoholic fermentation (beer, wine, champagne, martini, and sherry) on gastric acid output and release of gastrin was studied. Gastric acid output was determined by the method of intragastric titration. Plasma gastrin was measured using a specific radioimmunoassay. RESULTS: None of the alcoholic beverages produced by fermentation plus distillation had any significant effect on gastric acid output and release of gastrin compared with control (isotonic glucose and distilled water). Alcoholic beverages produced only by fermentation significantly (p < 0.05) increased the gastric acid output by 57% to 95% of maximal acid output (MAO) and release of gastrin up to 5.1-fold compared with control. If beer, wine, and sherry were distilled, only their remaining parts increased gastric acid output by 53% to 76% of MAO and increased release of gastrin up to 4.3-fold compared with control. CONCLUSIONS: (1) Alcoholic beverages produced by fermentation but not by distillation are powerful stimulants of gastric acid output and release of gastrin; (2) the alcoholic beverage constituents that stimulate gastric acid output and release of gastrin are most probably produced during the process of fermentation and removed during the following process of distillation.
PMCID: PMC1027007  PMID: 9155575
22.  Intensive care. 
Postgraduate Medical Journal  1993;69(811):340-358.
PMCID: PMC2399818  PMID: 8346129
23.  Randomised trial of safety and efficacy of immediate postoperative enteral feeding in patients undergoing gastrointestinal resection. 
BMJ : British Medical Journal  1996;312(7035):869-871.
OBJECTIVES: To assess whether immediate post-operative enteral feeding in patients who have undergone gastrointestinal resection is safe and effective. DESIGN: Randomised trial of immediate post-operative enteral feeding through a nasojejunal tube v conventional postoperative intravenous fluids until the reintroduction of normal diet. SETTING: Teaching hospitals in London. SUBJECTS: 30 patients under the care of the participating consultant surgeon who were undergoing elective laparotomies with a view to gastrointestinal resection for quiescent, chronic gastrointestinal disease. Two patients did not proceed to resection. MAIN OUTCOME MEASURES: Nutritional state, nutritional intake and nitrogen balance, gut mucosal permeability measured by lactulose-mannitol differential sugar absorption test, complications, and outcome. RESULTS: Successful immediate enteral feeding was established in all 14 patients with a mean (SD) daily intake of 6.78 (1.57)MJ (1622 (375) kcal before reintroduction of oral diet compared with 1.58 (0.14) MJ (377 (34) kcal) for those on intravenous fluids (P < 0.0001). Urinary nitrogen balance on the first postoperative day was negative in those on intravenous fluids but positive in all 14 enterally fed patients (mean (SD) - 13.2 (11.6) g v 5.3 (2.7) g; P < 0.005). There was no difference by day 5. There was no change in gut mucosal permeability in the enterally fed group but a significant increase from the test ratios seen before the operation in those on intravenous fluids (0.11(0.06) v 0.15 (0.12); P < 0.005). There were also fewer postoperative complications in the enterally fed group (P < 0.005). CONCLUSIONS: Immediate postoperative enteral feeding in patients undergoing intestinal resection seems to be safe, prevents an increase in gut mucosal permeability, and produces a positive nitrogen balance.
PMCID: PMC2350561  PMID: 8611872
24.  DNA methylation associated with repeat-induced point mutation in Neurospora crassa. 
Molecular and Cellular Biology  1995;15(10):5586-5597.
Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated amRIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.
PMCID: PMC230809  PMID: 7565710
25.  Community participation in health care decision making: is it feasible? 
Health care reform strategies proposed by provincial governments include decentralized funding and increased public participation in decision making. These proposals do not give details as to the public participation process, and a number of questions have been raised by the experience of some communities. Which citizens should form the decision-making group? What information do they need? What kinds of decisions should they make? What level of participation should they have? The results of a survey by Abelson and associates (see pages 403 to 412 of this issue) challenge the assumption that "communities" are willing to participate in health-care and social-service decision making. Willingness varied according to the composition of the groups polled, and participants' support for traditional decision makers increased after the complexities of the decision-making process were discussed. However, whereas their study measured willingness to participate at one point in time only, experience gained from Ontario's Better Beginnings, Better Futures project indicates that, given sufficient time, "ordinary" citizens are willing and can acquire the skills needed to decide how resources should be allocated for social services.
PMCID: PMC1487233  PMID: 7634219

Results 1-25 (65)