Previous studies have shown that atherosclerosis of the descending aorta detected by transesophageal echocardiography (TEE) is a good marker of coexisting coronary artery disease. The aim of our study was to evaluate whether the presence of atherosclerosis on the descending aorta during TEE has any prognostic impact in predicting cardiovascular events.The study group consisted of 238 consecutive in-hospital patients referred for TEE testing  with a follow up of 24 months. The atherosclerotic lesions of the descending aorta were scored from 0 (no atherosclerosis) to 3 (plaque >5 mm and/or "complex" plaque with ulcerated or mobile parts).Atherosclerosis was observed in 102 patients,  whereas 136 patients only had an intimal thickening or normal intimal surface. There were 57 cardiovascular events in the follow-up period. The number of events was higher in the 102 patients with (n = 34) than in the 136 patients without atherosclerosis . The frequency of events was in close correlation with the severity of the atherosclerosis of the descending aorta. Fifty percent of the patients with grade 3 experienced cardiovascular events. Excluding patients with subsequent revascularization, the multivariate analysis only left ventricular function with EF < 40%  and TEE atherosclerotic plaque >=2  predicted hard cardiovascular events.Atherosclerosis of the descending aorta observed during transesophageal echocardiography is a useful predictor of cardiovascular events. During the past decades many methods and factors have been proposed as good prognostic tools or markers for cardiovascular risk stratification. However, even with the most sophisticated stress testing procedures the prediction capability still remains imperfect . Previound Department of Medicine and Cardiology Center, University of Sciences, Szeged, Hungary. The patients underwent TEE examination for the following reasons: TIA or suspected cerebral embolism (n = 100), coronary flow reserve evaluation (n = 71), evaluation of the native or artificial mitral valve (n = 23), suspected endocarditis (n = 15), evaluation of the aortic valve (n = 13), suspected aortic dissection (n = 6), atrial septal defect or patent foramen ovale (n = 6), others (n = 4). Thirty patients had suffered a previous myocardial infarction. The patients were followed-up to a period of at least 2 years, median 31 ± 9 months.During the year 1998, 238 consecutive patients  were studied with transesophageal echocardiography at the 2All patients had a transthoracic echocardiographic examination to assess the global and regional left ventricular function. The ejection fraction was calculated using the area-length single plane method . Left veThe TEE examination was carried out according to recommendations of the Mayo clinic procedure . Two-dimGrade 0 – no sign of atherosclerosisGrade 1 – intimal thickeningGrade 2 – plaque < 5 mmGrade 3 – plaque > 5 mm and/or "complex" plaque with ulcerated or mobile parts.Significant atherosclerosis was considered in case of grade 2 or 3 fig , 2, 3, 4Follow-up data were obtained from at least one of four sources: 1) review of the patient's hospital record; 2) personal communication with the patient's physician and review of the patient's chart; 3) a telephone interview with the patient conducted by trained personnel; or 4) a staff physician visiting the patients at regular intervals in the outpatient clinic. Follow-up data were obtained in all patients. The outcome events were: cardiac related death, fatal stroke, non-fatal myocardial infarction and revascularization either by percutaneous transluminal coronary angioplasty (PTCA) or by coronary artery by-pass grafting (CABG). Myocardial infarction was documented by a consistent history, EKG changes and cardiac enzyme level elevations and confirmed by hospital chart or hospital discharge letter review. In the next step, patients with revascularization were censored and the remaining patients were considered for the analysis of hard events . All-cause mortality (cardiovascular death plus death of other causes) was also analyzed.Values are expressed as mean ± Standard Deviation. Continuous variables have been compared by the means of Student's t test (two-tailed). Statistical analysis of discrete variables has been performed with chi-square test; a Fisher's exact test has been used when appropriate. The individual effect of certain variables on infarction-free survival has been evaluated with the use of the Cox proportional hazard model with univariate and multivariate analysis and with the Kaplan-Meier method. The patients were stratified into two subgroups: patients with and without cardiac events. The examined variables were age, sex, risk factors , previous myocardial infarction, ejection fraction, and significant atherosclerosis of the descending aorta. A p value <0.05 was considered statistically significant.The examination was successful and complete in all patients, without any side effect. Significant atherosclerosis of the descending aorta was observed in 102 patients,  whereas 136 patients had only mild intimal thickening (n = 46) or normal endocardial surface (n = 90).There were 57 events in the follow-up period: cardiac related death: n = 14, fatal stroke: n = 4, non-fatal myocardial infarction: n = 5 and coronary artery revascularization: n = 34. Ten patients died of non-cardiovascular causes and the cause of the death of 2 patients was undetermined.The number of events was significantly higher in the 102 patients with (n = 34) as in patients the 136 patients without significant atherosclerosis . The results of univariate and multivariate analysis are shown in table The frequency of events was in close correlation with the severity of the atherosclerosis of the descending aorta fig . Fifty pPatients with early  or late  revascularization have been censored.Nine events have occurred in the 122 patients with TEE score ≤1 and 14 in the 82 patients with TEE score ≥2  fig . The resAtherosclerosis of the descending aorta observed during transesophageal echocardiography is a useful predictor of future cardiovascular events.Our data are in keeping up with previous findings, showing that patients with atherosclerosis of the thoracic aorta have higher probability of coexisting coronary artery disease -8. In thOne of the most important first steps in stratifying risk among patients with proven or suspected coronary artery disease is the identification of patients at high risk for coronary or vascular events during the course of the next few months or years. To date, left ventricular dysfunction, the number of diseased vessels, and the severity of myocardial ischemia have emerged as important determinants of survival . Our datThis study has several limitations. The study population was highly heterogeneous, reflecting the garden variety of patients referred to the echo lab for transesophageal testing: patients with known or suspected coronary artery disease or cerebrovascular disease coexist with patients with congenital or acquired valvular disease. Therefore, our findings cannot be directly translated into the general population, and further prospective studies are needed at this point to evaluate the prognostic value of transesophageally detected aortic pathology in more sharply defined clinical subsets.The morphological characterization of atherosclerotic plaque has not been performed in our study, neither in terms of plaque content  nor of pGrade I. atherosclerosis of the descending aorta. Intimal thickening.Click here for fileSmall plaque on the descending aorta, corresponding to Grade 2. atherosclerosis.Click here for fileGrade 3. atherosclerosis, with large, multiple plaques.Click here for fileGrade 3. atherosclerosis, plaque with mobile parts.Click here for file


An Outbreak Control Team was formed resulting in extensive epidemiological, microbiological and environmental investigations.On 30th July and a standard case definition was adopted. Using a pre-designed proforma information was gathered from both sick and well subjects. Food specific attack rates were calculated and two-tailed Fisher's exact test was used to test the difference between type of food consumed and the health status. Using a retrospective cohort design univariate Relative Risks and 95% Confidence Intervals were calculated for specific food items.Attempts were made to contact everybody who ate food from the restaurant on 27S Enteritidis phage type 34a and one for S Enteritidis phage type 4.Data was gathered on 52 people of whom 38 developed gastrointestinal symptoms; 16 male and 22 female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours. Food attack rates were significantly higher for egg, special and chicken fried rice. Relative risk and the Confidence interval for these food items were 1.97 (1.11–3.48), 1.56 (1.23–1.97) and 1.48 (1.20–1.83) respectively. Interviews with the chef revealed that many eggs were used in the preparation of egg-fried rice, which was left at room temperature for seven hours and was used in the preparation of the other two rice dishes. Of the 31 submitted stool specimens 28 tested positive for In the absence of left over food available for microbiological examination, epidemiological investigation strongly suggested the eggs used in the preparation of the egg-fried rice as the vehicle for this outbreak. This investigation highlights the importance of safe practices in cooking and handling of eggs in restaurants. S Enteritidis, S Typhimurium and S Virchow [Infection due to Salmonella is a major public health problem in England and Wales with reports of over 14,400 infections due to Salmonella in the year 2003 [Salmonella enterica serovar Enteritidis phage types 4, 21 and 6 have been reported in previous outbreaks [S Enteritidis PT 34a infection reported in the literature from United Kingdom. In this report, we present the results of an epidemiological investigation of an outbreak due to this rare phage type associated with a Chinese restaurant in Suffolk, United Kingdom.Although utbreaks , phage tth July 2002, the Suffolk Communicable Disease Control team (SCDC) was informed by the consultant microbiologist that S. Enteritidis had been isolated from stool samples of five patients. All had recently eaten a meal in a local Chinese restaurant. Further enquires revealed that there were more patients with similar food history and gastrointestinal symptoms. An Outbreak Control Team was convened on 31st July and it was decided that a full investigation should be carried out to identify the extent of the outbreak, the probable vehicle of infection and to advise on the appropriate control measures.On 30th July 2002. The information collected included name, address, sex, their symptoms and date of onset. The restaurant provided the list of food items that were served/sold on the day in question. This menu extended to 40 food items. This list was shown to the restaurant patrons and they were asked to state the food items they had eaten. A variety of ways was used to identify further cases including the technique of snowball sampling. This involved asking the patrons whether they were aware of any others who had similar symptoms and had eaten in the restaurant. General Practitioners providing primary care in the area were contacted and were requested to check for patients with gastrointestinal symptoms. The presenting symptoms of the patrons were diarrhoea, headache, abdominal pain and fever.The environmental health department (EHD) staff initially gathered information from people who had become ill on a standard data collection form. In the initial stages of the investigation, it became apparent that all those who became ill had eaten or had bought a take away at the restaurant on 27th July whether they became ill or not. The restaurant provided table-booking details. The following case definition was adopted for the outbreak. "Symptoms of acute gastroenteritis including one of the following: diarrhoea, vomiting or abdominal pain up to 96 hrs after having had a meal from the said restaurant including takeaway between 22 and 30 July 2002 and/or individuals who have positive stool sample for S. Enteritidis up to 96 hrs after having a meal from the restaurant including a takeaway between 22 and 30 July 2002".The next step involved interviewing all those who had eaten/bought food on 27th July. Eligibility for membership of the cohort was defined as a person having the opportunity to eat any of the food items available on the day.An analytical investigation was carried out using a retrospective cohort design. Efforts were made to identify anyone who ate or bought food at the restaurant on 27Data was entered in to Statistical Package of Social Sciences version 10 . Food spth of July. Environmental sampling was not carried out as this was considered to be of limited value. There was no food left over from 27th July, but three food samples were taken on 30th July and sent for analysis to the food laboratory at Chelmsford Public Health Laboratory. Stool specimens were sent to Ipswich Hospital microbiology laboratory and were cultured for the presence of Salmonella sp. Isolates of Salmonella were forwarded to the Laboratory of Enteric Pathogens at Central Public Health Laboratory, Colindale for phage typing. Standard procedures were adopted for phage typing at the laboratory [Stool samples were requested from all who had eaten food from the restaurant on 27boratory .The EHD staff inspected the premises including verifying the procedures for hazard analysis and critical control point (HACCP). Egg storage and preparation of egg items were also investigated during the visit. Efforts were made to trace the egg trail back to the supplier.th July of whom 38 developed symptoms and 14 were free of symptoms. Of the 38 who became ill 16 were male and 22 were female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours suggesting a point source outbreak. Two patients received hospital care and there were no deaths. No gastrointestinal illness was reported among the kitchen staff of the restaurant in the weeks before or during the outbreak.Data were gathered from 52 subjects who had eaten food from the restaurant on 27On investigation of food preparation practices at the restaurant, it appeared dishes containing egg were the most likely vehicle for this outbreak. However, this information was not discussed when gathering data from the subjects. Data was gathered in a standardised format from all subjects to avoid any interviewer or recall bias.We had a strong "a priori" hypothesis that people who had eaten egg or food that had come in to contact with egg were at an increased risk even before looking at the data and these were analysed first. During analysis it became apparent that illness was significantly associated with the three food items that contained egg or the egg rice mixture as shown by the food specific attack rates Table  and the S. Enteritidis was isolated in 29. Twenty-eight of these 29 isolates were confirmed to be PT 34a and one as PT4. No pathogens were isolated from food samples taken from the restaurant on 30th July.A total of 31 stool specimens were submitted to the laboratory from which th of July the egg rice mixture was left at room temperature for seven hours. The restaurant received eggs from a supplier in London every week and they were not refrigerated. Attempts to trace the egg trail were not successful.During the visit to the restaurant, the EHD staff reported that there was no evidence of hazard analysis and noted many cleaning and maintenance issues. Hand wash facilities were inadequate. The chef explained that after preparing the egg rice mixture, it was left out at room temperature for the rest of the evening, and reheated when ordered. This mixture was used for some of the other fried rice items. It was estimated that on the evening of the 27st July and the EHD staff reassessed the situation on the evening of 1st of August. As they were satisfied with the arrangements, the restaurant was allowed to reopen. The owner and restaurant staff were provided with information on proper cooking methods and the importance of undertaking HACCP.The restaurant closed voluntarily on 31The epidemiological investigation showed that eggs used in the preparation of egg-fried rice, which in turn was used in the preparation of some of the other rice items was the vehicle of infection. Isolation of the unusual phage type 34a strengthened the conclusion that eating from the restaurant was linked to a point source outbreak. In general, by the time investigations are initiated often no food material is available for laboratory analysis and the investigator has to rely on epidemiological evidence. The first step in identifying the source of an outbreak is the calculation of attack rates and the responsible food should have a significantly higher attack rate . In thisStatistical methods have better power while there is an "a priori" hypothesis as shown in the study of summer excess of leukaemia . We had S. Enteritidis outbreaks associated with Chinese food businesses in England [To our knowledge, this is the third report of an outbreak due to phage type 34a and the first of its kind in England published in the literature. In the UK, this phage type has been associated with travel abroad especially to southern Spain  and indi England , Scotlan England  and the  England  although England , egg rolS. Enteritidis has been reported to be low [S. Enteritidis in causing food borne outbreaks is well known as it has the ability to contaminate eggs without causing discernible illness in the birds affected [et al [The proportion of eggs infected with o be low  and hencaffected . Eggs haaffected -22. Hayed [et al  in theirS. Enteritidis phage types appear to be genetically related or clonal. Discussion on stability of phage types of S. Enteritidis can also be found in the literature. Conversion of phage type 4 to 24, phage type 23 to 8 and 4 to 7 have been reported. We could not find reports linking phage type 34a and phage type 4. In a recent public health investigation [S. Enteritidis in raw eggshells, various serotypes of Salmonella were isolated from 23 out of 449 (5.1%) pooled samples labelled as originating from Spain. These sero/phage types included S. Enteritidis PT6a, PT5c, 13a, 14b, 58, PT6d, PT1, PT1c and PT12.We could not find any veterinary data on phage type 34 in British flocks. We searched the literature to determine whether there is any molecular relationship between phage type 34a and phage type 4. Hudson et al  based ontigation  of S. EnA few limitations of this study are to be noted. The origin of the suspected contaminated eggs could not be traced. Although trace back exercises are key in epidemic investigations, often they are not successful due to logistic and practical reasons. In our outbreak one of the suppliers to the restaurant turned out to be a packaging firm. During our investigation, we found that there were problems with the distribution system, which prevented us from pin pointing the origin of the contaminated eggs. A possibility always exists that we missed a few subjects from this investigation and not all could be persuaded to provide a stool sample. There was no single list of all the patrons who ate/purchased food on the evening. However, all efforts were made to contact the patrons and the outbreak caused considerable publicity in the local media. Hence, we are confident that we have included most patrons. Although the precise number of patrons who were not included will never be known we are confident that their number is small and might be in the region of 10 to 15.Palmer  has poinS. Enteritidis PT4 was isolated from one of the subjects. Further investigation revealed that this subject had recently returned from holiday in continental Europe and had suffered mild symptoms before the meal.In response to this and other outbreaks associated with eggs, a Public Health Investigation was launched in October 2002 in the UK to determine the rate of Salmonella contamination in eggs. Tests of nearly 4000 eggs showed that Salmonella was recovered from 5.3% of pooled eggs . The FooInvestigation of this outbreak was greatly facilitated by the close cooperation between local EHD, Communicable Disease Control Team, microbiological laboratory and local health care providers. Although food samples from this point source outbreak were not available for microbiological culture, epidemiological evidence pointed to eggs containing dishes as the most likely source of the outbreak. This outbreak highlights the continuing hazards of raw eggs. It is likely that the use of pasteurised eggs and the adoption of safe food preparation practices would have prevented this outbreak.The first author (PB) served as the expert witness for the prosecution during the court proceedings.PB and TS conceived and designed the study and drafted the manuscript.PB analysed the dataRK oversaw the microbiological investigation.PB, TS, RK and HM all interpreted the results of the analysis and critically reviewed the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Tertiary care hospitals are a potential source for development and spread of bacterial resistance being in the loop to receive outpatients and referrals from community nursing homes and hospitals. The liberal use of third-generation cephalosporins (3GCs) in these hospitals has been associated with the emergence of extended-spectrum beta- lactamases (ESBLs) presenting concerns for bacterial resistance in therapeutics. We studied the 3GC utilization in a tertiary care teaching hospital, in warded patients  prescribed these drugs.Clinical data of patients (≥ 13 years) admitted to the General Hospital, Port of Spain (POSGH) from January to June 2000, and who had received 3GCs based on the Pharmacy records were studied. The Sanford Antibiotic Guide 2000, was used to determine appropriateness of therapy. The agency which procures drugs for the Ministry of Health supplied the cost of drugs.The prevalence rate of use of 3GCs was 9.5 per 1000 admissions and was higher in surgical and gynecological admissions (21/1000) compared with medical and orthopedic (8 /1000) services (p < 0.05). Ceftriaxone was the most frequently used 3GC. Sixty-nine (36%) patients without clinical evidence of infection received 3Gcs and prescribing was based on therapeutic recommendations in 4% of patients. At least 62% of all prescriptions were inappropriate with significant associations for patients from gynaecology (p < 0.003), empirical prescribing (p < 0.48), patients with undetermined infection sites (p < 0.007), and for single drug use compared with multiple antibiotics (p < 0.001). Treatment was twice as costly when prescribing was inappropriateThere is extensive inappropriate 3GC utilization in tertiary care in Trinidad. We recommend hospital laboratories undertake continuous surveillance of antibiotic resistance patterns so that appropriate changes in prescribing guidelines can be developed and implemented. Though guidelines for rational antibiotic use were developed they have not been re-visited or encouraged, suggesting urgent antibiotic review of the hospital formulary and instituting an infection control team. Monitoring antibiotic use with microbiology laboratory support can promote rational drug utilization, cut costs, halt inappropriate 3GC prescribing, and delay the emergence of resistant organisms. An ongoing antibiotic peer audit is suggested. Appropriate use of antibiotics is central to limiting the development and the spread of resistant bacteria in hospitals and communities. Use of broad-spectrum antibiotics, in particular the 3GCs in nosocomial infections have been linked to the emergence of antibiotic resistance and increase in costs . The hosEscherichia coli and Klebsiella spp. An important source of this resistance results from the production of extended-spectrum beta-lactamases (ESBLs) by bacteria. ESBLs are modified beta-lactamase enzymes mainly derived from the ubiquitous TEM1/2 and SHV-1 plasmid-mediated enzymes, which hydrolyse expanded spectrum cephalosporins to varying degrees. Many beta-lactamases result in resistance to 3GCs in Enterobacteriaceae. Genera such as Enterobacter, Citrobacter and Serratia posses chromosomal broad spectrum beta-lactamases which are normally repressed, and when induced result in resistance to 3GCs. Klebsiella and E. coli usually have the SHV- or Tem- type beta-lactamases, and key mutations in these result in true "ESBLs". ESBLs have received attention in the last decade because although penicillins, cephalosporins, or aztreonam appear to be susceptible in vitro, ESBL producing E. coli or Klebsiella spp. may demonstrate clinical resistance to these antibiotics leading to treatment failures. Liberal use of the 3GC antibiotics has resulted in the ESBLs conferring resistance among Enterobacter[K pneumoniae and other Enterobacteriaceae and vancomycin-resistant enteroccocci with cephalosporin use [Enterobacteriaceae has been reported from Barbados [Enterobacteriaceae has recently been observed in tertiary care in Jamaica [The increasing resistance to 3GCs accompanied by an increasing cost burden has raised concerns about the detection, prevalence, and clinical implications of infections with erobacter-6 comproorin use -11. ReceBarbados , and ext Jamaica  and Trin Jamaica . From unIn Trinidad and Tobago, health care is delivered through the public sector, though several private facilities are also available. At the publicly-financed health care institutes patients undergo investigations and receive treatment without cost. There are 2 tertiary general hospitals in Trinidad, the General Hospitals at Port of Spain and San Fernando and several secondary and primary health care facilities where patients can access medical care. The POSGH is the country's major health care institute and receives referrals from all over the country including the sister isle of Tobago. This hospital is a 900-bed institute providing out-patient services and health care for warded patients for over one third of Trinidad and Tobago's population of 1.3 million. Medical students undergoing training at the University of the West Indies, also attend clinical rotations at this hospital.Between January to June 2000, we conducted a cross sectional study of adult in- patients (over 13 years) who had received one or more courses of treatment with one of the 3GCs  available at the POSGH. The Hospital Pharmacy identified warded patients from the respective services based on the prescriptions dispensed. Patient characteristics, clinical data and laboratory investigations were obtained from the hospital records. While we did consider the course of antibiotic duration in defining appropriateness of therapy, we did not analyse data based on use per patient days because these data were not clearly available from the records. Specific data on the category of service, concomitant disease and drug therapy, organ system with infection, invasive/indwelling devices and the 3GC used were collected using a standardized instrument.An infection was deemed to be present when the physician's diagnosis of infection was a differential diagnosis stated in the chart and/or in a patient who had a fever (>100.4 C) and elevated WBC Count >12,000/cmm.On review all patients with diabetes and HIV had a clinical diagnosis of infection stated in the records.The appropriateness of antibiotic therapy was determined using the criteria described in the Sanford Antibiotic Guide, 2000. Therapy was deemed to be inappropriate based on any of the following parameters: the type of therapy (prophylactic and empiric), combination of antibiotics, the route of administration, the dose, and the duration of therapy. The Sanford guide is widely accepted in Trinidad and the Caribbean as a reference for making appropriate therapeutic recommendations and is also used as a reference manual by medical microbiologists. No other guidelines are currently available in Trinidad. The course of therapy was considered a parameter of appropriate therapy, but a separate analysis based on this parameter was not possible since the information was not always available from the patient records.The direct cost of total antibiotic treatment was computed using the data obtained from the National Insurance Property Development Corporation, which is the agency contracted by the Ministry of Health for procurement and distribution of drugs in the public sector health institutes.2) tests.Data were analysed using EPI Info 6.4  and categorical variables were compared using the Odds Ratio (95% CI) and the chi-square (XOne hundred and ninety two (192) adult patients admitted to the POSGH were treated with 3GCs during the first six months of 2000, providing a prevalence rate of use of 9.5 patients per 1000 admissions, (192/20146). The rate of use was higher (p < 0.05) in those patients utilizing the surgical and gynecological services (21 per 1000 admissions) compared with those admitted in the medical and orthopedic services (8 per 1000 admissions). One hundred and twenty seven (66%) patients received ceftriaxone, cefotaxime was prescribed for 51 (26.5%) patients, and ceftazdime was administered to 14 (7.5%) patients.The demographic characteristics of patients who received a 3GC and factors related to infection and treatment in the study population are shown in Table There were 107(56%) patients who had fever and a high WBC count with leucocytosis and who were considered to have a clinically suspected infection. Thirty six percent of patients who had no clinical evidence of infection had received treatment with a 3GC. The most common site of suspected infection (20.3%) was the skin and soft tissue followed by the respiratory (10.9 %), gastrointestinal (10.7%), and urinary (9.3%) tracts.Sixty eight percent (131) of patients in this study received empirical antibiotic treatment and in 29.7% of patients these antibiotics were prescribed as prophylactic therapy. Only 4% of patients received 3GCs based on recommended therapeutic regimens. More patients (87%) who were admitted to the medical services received empirical antibiotic treatment compared with patients in the surgical services (68%), (p < 0.05). Prophylactic antibiotic regimens were most frequently prescribed in the orthopedic (50%) and gynecological services (45%) in contrast to patients in surgery (31%) who received 3GCs as prophylactic therapy. However patients in the surgical units (61%) were more likely to be administered two or more antibiotics compared with those receiving medical care in the orthopedic (33%) or gynecological (45%) services (p < 0.01). Single antibiotic therapy was prevalent in the medical, orthopedic, and gynecological services . Seventy-eight percent of those patients who did not display any clinical evidence of infection were treated with one antibiotic. Patients with infections of the skin and soft tissue (75%) and the urinary tract (61%) were more likely to receive treatment with two or more antibiotics compared with patients admitted with respiratory tract infections (48%) (p < 0.05).Pseudomonas spp (7), Acinetobacter spp (3), Enterobacter spp (2), Klebsiella spp (2), Proteus (2) S. aureus (3), and others (8).Biological samples were not sent to the laboratory in as many as 73% of patients to determine a bacteriological etiology of suspected infection. Table . Of the An analysis of factors that were associated with the inappropriate use of 3GCs in the patient sample is shown in Table The average duration of patient stay in the hospital was 14 ± 22 days (range 1 day -150 days). Patients in the orthopedic wards were hospitalized for more days (24.5 ± SD 35.28) compared with the number of days patients spent in the medical (12.45), surgical (13.7) and gynecology (7 days) services. Interestingly, the mean duration of stay of patients in whom antibiotic treatment was appropriate, was not significantly different from the number of days that patients who were treated inappropriately were warded. The overall direct costs from use of these antibiotics in this study was TT$ 117,432 (US$ 19251.00). The cost of treatment with these antibiotics in patients who were inappropriately treated was  and was twice as high as the cost of treatment for those patients who were treated appropriately The extensive use of third generation cephalosporin antibiotics has caused the emergence of extended spectrum beta-lactamases in Gram-negative bacteria worldwide -6. More In our study, the prevalence rate of use of 3GCs was lower than the reported rate of 43 per 1000 admissions for ceftriaxone in 51 Victorian Hospitals in Australia of which 82% was prescribed as empirical treatment . Our datEnterobacter spp to cefotaxime and ceftriaxone was 53% and 37% respectively in the Intensive Care Unit at this hospital. It is interesting that only 4% of prescriptions were based on therapeutic regimen following culture and sensitivity results in our study, and in 73% of patients with suspected infection bacteriological confirmatory tests were never done and 3GCs were prescribed. We believe high inappropriate use of these antibiotics has contributed to increasing multiple antibiotic resistance and extended spectrum beta-lactamase producing enterobacteriaceae observed in this hospital [Third generation cephalosporins were introduced in the Caribbean between the late eighties and the early nineties and soon after in 1993, the first isolation of resistant Gram-negative bacilli resistant to them was demonstrated in Barbados . In Jamahospital .The cost of inappropriate antibiotic use was twice as much for patients who were treated appropriately and highlights irrational antibiotic consumption at the hospital, prompting protocols for rational antibiotic prescribing and utilization review. The World Health Organization has recommended multifaceted strategies to improve hospital-prescribing practices such as the development of consensus guidelines, educational activities and rapid feedback to prescribers about inappropriate use to reduce overuse of antibiotics in the hospital . RestricThis first audit on 3GC use from the Caribbean demonstrates inappropriate use of these drugs in tertiary care and presents opportunities to develop consensus guidelines for rational use of these drugs in hospitals. We recommend microbiological services in the hospitals undertake continuous surveillance of resistance patterns, to guide in the development of prescribing guidelines. Such guidelines should be widely disseminated and implemented in these institutions, so that prescribers remain informed of rational therapeutics. This study highlights the need to constitute an antibiotic monitoring team comprising a pharmacist, physician, medical microbiologist and infection control nurse to periodically review and evaluate the use and cost of antibiotics at the major tertiary care hospital in Trinidad, to assist clinicians in optimizing clinical care of patients with Gram-negative infections.3GCs Third generation cephaosporinsESBLs Extended spectrum Beta lactamsesPOSGH General Hospital, Port of SpainThe author(s) declare that they have no competing interests.LMPP conceived the study, the design, co-ordinated it and prepared the final manuscript. MP, HR and KT did the data collection. PP assisted with the co-ordination, did the analysis and prepared the draft manuscript. All authors saw the final manuscript and made contributions.The pre-publication history for this paper can be accessed here:


Nicotinic acid is a unique cholesterol modifying agent that exerts favorable effects on all cholesterol parameters. It holds promise as one of the main pharmacological agents to treat mixed dyslipidemia in metabolic syndrome and diabetic patients. The use of nicotinic acid has always been haunted with concerns that it might worsen insulin resistance and complicate diabetes management.We will discuss the interaction between phosphorous metabolism and carbohydrate metabolism and the possibility that worsening of insulin resistance could be related to adrug induced alteration in phosphorous metabolism, and the implications of that in medical management of diabetes and metabolic syndrome patients with mixed dyslipidemia. Nicotinic acid functions in the body after conversion to nicotinamide adenine dinucleotide (NAD) in the NAD coenzyme system. Niacin reduces total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), apolipoprotein B-100 (Apo B) and Lipoprotein (a) (Lp (a)). Niacin increases high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (Apo A-I), lipoprotein A-I, and HDL2: HDL3 ratio. The severity and type of underlying lipid abnormality determines the extent of response to niacin treatment [There is evidence suggesting that niacin reduces the risk of a coronary artery or cerebrovascular disease in the setting of secondary prevention, and when combined with a bile acid-sequestrant, promotes regression and decreases progression of atherosclerotic cardiovascular disease ,3.Niacin is known to cause transient, small but statistically significant dose-related reductions in phosphorous levels. These effects were studied for extended release niacin in placebo-controlled trials, mean percentage changes from baseline (± standard error) in phosphorous were -4.0 ± 1.3) for the 500 mg once daily dose, (-7.2 ± 1.1) for the 1000 mg once daily dose, (-9.1 ± 1.1) for the 1500 mg once daily dose, and (-12.6 ± 1.6) for the 2000 mg once daily dose [.0 ± 1.3 It has been long known that niacin induces impaired fasting glucose. I was also suggested that this effect is most likely transient. The mechanism behind this phenomenon has not been fully elucidated .Multiple studies were conducted to study effects of nicotinic acid on exacerbating insulin resistance. One retrospective study suggested that the use of moderate doses of extended release nicotinic acid  in reasonably controlled diabetics was associated with improved glycemic control with HbA1c levels decreased by 0.5% ± 0.3% due to aggressive hypoglycemic therapy; as most of these patients had their insulin or oral diabetes regimen intensified . AnotherThe (ADMIT) trial, was one of the first studies to demonstrate the safety of nicotinic acid in patients with diabetes. Diabetic patients  were randomized to treatment with either placebo or niacin (2500 mg/d) for 18 weeks. Patients randomized to niacin therapy had modest increases in fasting glucose (+8 mg/dL). There was no significant change in HbA1c levels or in diabetes treatment regimen in the niacin-treated diabetic patients group .It appears from the clinical data presented above, that nicotinic acid at higher doses could results in alterations in glycemic control of patients with an already abnormal glucose metabolism.Phosphate is primarily an intracellular anion. Insulin is implicated in the transport of glucose and phosphate from the intra-cellular to the extra-cellular space. Studies have suggested a link between decreased serum phosphorous and disturbed carbohydrate metabolism, eventually leading to hyperglycemia ,11. ChroIt seems intuitive to suspect that the transient decrease in plasma phosphorous level might explain the transient disturbance in carbohydrate metabolism and hyperglycemia induced by nicotinic acid. Also, studies suggest the existence of a dose response relationship in both instances.One way to test this hypothesis will be to administer nicotinic acid to both healthy subjects, and to those with impaired fasting glucose or subjects with diabetes, then measure simultaneously at different doses serum phosphorous, and correlate the changes to changes in serum glucose and serum insulin either individually or combined as a measure of insulin resistance.It will be of interest to study if such a relationship exists in patients with the metabolic syndrome diagnosis, since literature suggests the existence of an abnormal phosphorous metabolism in these patients ,12.This hypothesis has several important insights and implications:1. If valid, it suggests the possibility of a simple clinical way to prevent nicotinic acid induced worsening of glycemic control by dietary phosphorous supplementation, eliminating the need to intensify expensive diabetic regimens in diabetic patients that need nicotinic acid treatment.2. If valid, it will aid in lessening the theoretical anxiety of the possibility of precipitating diabetes in glucose intolerant or metabolic syndrome patients in need of nicotinic acid treatment for the management of dyslipidemia.3. Metabolic syndrome and diabetics stand to benefit greatly from the new evidence implicating the importance of increasing HDL-cholesterol in cardiovascular event reduction.4. This hypothesis offers new insights into the mechanism of development of glucose intolerance.


One of the most remarkable capabilities of the adult brain is its ability to learn and continuously adapt to an ever-changing environment. While many studies have documented how learning improves the perception and identification of visual stimuli, relatively little is known about how it modifies the underlying neural mechanisms. We trained monkeys to identify natural images that were degraded by interpolation with visual noise. We found that learning led to an improvement in monkeys' ability to identify these indeterminate visual stimuli. We link this behavioral improvement to a learning-dependent increase in the amount of information communicated by V4 neurons. This increase was mediated by a specific enhancement in neural activity. Our results reveal a mechanism by which learning increases the amount of information that V4 neurons are able to extract from the visual environment. This suggests that V4 plays a key role in resolving indeterminate visual inputs by coordinated interaction between bottom-up and top-down processing streams. In monkeys trained to identify natural images embedded in noise, changes are seen in the information about learned stimuli signaled by V4 neurons It is well established that learning can have a strong impact on neural responses to visual stimuli in high-level association cortices such as inferior temporal (IT) or prefrontal (PF) cortex, where the activity of single neurons reflects learning in pair association, object identification, or categorization tasks . In thesRecent evidence suggests that neurons in early visual sensory areas can also modify their response properties with learning. In particular, several studies have revealed learning-related changes in primary visual cortex (V1) , althougLearning thus appears to affect both low and high level areas of the ventral visual stream. The results obtained by studies in these two areas are, however, difficult to compare directly, owing to substantial differences in experimental design. In studies of IT or PF cortex, studies typically employ ‘complex' visual stimuli such as Fourier descriptors , computeFor a comprehensive account of how learning affects visual processing, the same stimuli and experimental procedure must be used to study different levels of the visual processing hierarchy. What kind of stimuli might be suitable to study visual areas as different as early sensory visual and PF cortex? We decided to use natural images for several reasons: The primate visual system evolved in the natural environment under conditions of ‘natural' stimulation; much is known about their statistical properties and they can therefore be well-controlled; they contain structure at all spatial scales and thus can be expected to activate a large fraction of visually responsive neurons. We avoid subjectively biasing our sample of recorded neurons by always recording from the first neurons whose waveforms we are able to reliably isolate. This ensures that our population of recorded neurons represents an unbiased sample in each brain region under study, and this in turn allows us to compare data obtained from different brain regions. We obtain a sensitive measure of behavioral performance and associated neural activity by employing a stimulus degradation procedure that makes stimuli harder to discriminate by adding various amounts of noise see A. With dHere our aim is to use similar stimuli and behavioral procedures to characterize how learning modifies neural activity in extrastriate visual cortical area V4. Area V4 was chosen because it is considered to be a sensory visual area at an intermediary processing stage in the ventral stream and because it is directly connected to parts of the PF cortex . Our tast-test, p < 0.01). For undegraded stimuli at 100% coherence, the monkeys' performance was near ceiling for both novel and familiar stimuli . Learning-dependent performance improvements for degraded stimuli were highly consistent across stimuli and monkeys. There were in fact no significant differences in the monkeys' performance to each of the familiar stimuli across sessions at all coherence levels , and this was also true for novel stimuli. In addition, performance for novel and familiar stimuli did not differ significantly between the two monkeys at any coherence level . Note that the monkeys' excellent perfor-mance with undegraded novel objects reflects the fact that they have acquired the rule of the DMS task and are thus able to perform it near ceiling with novel stimuli. The timecourse of this learning-dependent difference in performance is shown in t-test, p = 0.43). Furthermore, the learning-dependent difference in performance appeared to asymptote after around ten sessions. In summary, learning led to robust improvements in the monkeys' ability to identify degraded natural images while the monkeys performed near ceiling for novel and familiar undegraded images.We found that learning resulted in significant and robust improvements in monkeys' ability to identify degraded stimuli. Behavioral performance varied systematically with coherence A. Monkeynov〉 = 36.7 ± 2.8 Hz) and familiar stimuli (〈FRfam〉 = 34.2 ± 2.7 Hz) was similar , similar fractions preferred familiar or novel stimuli . We thus found that learning did not lead to a change in the average activity of V4 neurons for undegraded stimuli. Next, we examined whether learning resulted in any change in the amount of stimulus-specific information that V4 neurons communicated. To do this, we computed the mutual information between the set of four familiar or novel stimuli and the associated neural responses . We selected 25% of the population (21 out of 83 neurons), which communicated most information about novel or familiar objects . Thus, for natural images (undegraded stimuli) we saw no significant learning-dependent difference in performance and also no learning-dependent changes in the average activity or in the amount of stimulus-specific information communicated by V4 neurons.We now examine whether there were any learning-depen-dent changes in the activity of V4 neurons. Results described in this report are based on a population of 83 V4 neurons. We first asked whether there was any general difference in mean activity elicited by novel and familiar undegraded stimuli. We found that the response of V4 neurons to novel . For the 25% most informative neurons , corresponding to a 40% increase in information with learning. We further characterized this effect by examining how degradation affected the amount of information separately for novel and familiar stimuli. For both novel (100) than degraded (Idegrad) stimuli , reflecting the fact that behavioral perfor-mance was better for undegraded than degraded stimuli (see 100 − Idegrad) for familiar and novel stimuli shown in the insets , and learning was associated with a rightward shift in this distribution . Interestingly, the kurtosis or skewness of the ΔI distribution changed by an order of magnitude from 0.13 for novel stimuli to 5.5 for familiar stimuli, similar to experience-dependent effects that have been observed on hippocampal place cell activity . Taken together, learning accordingly resulted in an increase in the amount of information communicated by V4 neurons about degraded stimuli and many neurons actually communicated more information about degraded than undegraded familiar stimuli.At intermediate coherence levels, the monkeys' ability to correctly identify degraded stimuli was improved by learning, and we asked whether this behavioral improvement was associated with any changes in the activity of V4 neurons. We found that V4 neurons indeed communicated significantly more information about degraded familiar than about degraded novel stimuli B. Considth novel A and famth novel B stimulimuli see A. The ΔIe insets A and 4B,activity . As a cot-tests, p < 0.01). For this neuron . By contrast, activity to novel stimuli systematically decreased with degradation and was significantly below activity to undegraded stimuli at coherence levels of 35% and 45% . As expected, V4 neurons generally showed greater activity to novel and familiar stimuli than to pure noise at 0% coherence . As detailed in t-test, p < 0.05). Taken together, learning resulted in an increase in information communicated by V4 neurons about degraded or indeterminate stimuli. This increase in information was mediated by neurons that showed an enhancement in neural activity to degraded compared to undegraded familiar stimuli.How did single V4 neurons mediate this learning-depen-dent increase in information about degraded stimuli? The activity of an example neuron is shown in 2  for novel stimuli to 1.47 ± 0.16 dva2 for familiar stimuli . There were also significant learning-dependent increases in the high-probability FPM areas at 45% and 100% coherence . This learning-dependent increase in the high probability FPM regions and their overlap was highly consistent across sessions and monkeys, and we observed it during all six sessions in both monkeys. Note that the lower FPM values for novel stimuli indicate that eye position was less focused and therefore more distributed before learning, whereas for familiar stimuli robust regions of focused eye position developed.We performed additional behavioral experiments to assess whether learning led to any changes in fixational eye movements, because such changes might shed light on what mediates monkeys' behavioral advantages for familiar degraded stimuli. In these studies, we allowed the monkeys to freely view sample stimuli during task performance and then estimated a fixation probability map (FPM) for each familiar and novel stimulus presented at 45% and 100% coherence  levels with decreasing stimulus coherence in area V4 of anesthetized monkeys using novel stimuli  . After Our findings in V4 are in stark contrast to data obtained in the PF cortex using similar task and stimuli . In the The task dependence of learning effects in V1  has beenSeveral computational models have investigated how brain regions might interact during stimulus identification. A key feature of such models is the interaction between bottom-up and top-down processing . ConsideMacaca mulatta) participated in these experiments. All studies were approved by local authorities and were in full compliance with applicable guidelines (EUVD 86/609/EEC) for the care and use of laboratory animals. Stimuli were 10° × 10° in size, 24-bit color depth, and presented at the center of gaze on a γ-corrected 21-inch monitor with linear display characteristics placed at a distance of 97 cm from the monkeys. Stimuli were generated using Fourier techniques that have been described in detail elsewhere . After a delay of 1000 ms, a probe stimulus was presented for 600 ms, which could be any one of the eight undegraded images (100% coherence). The monkeys were required to release the lever if the probe matched the sample . In case of a nonmatch, a second brief delay (200 ms) followed the probe, and this delay was always terminated by the presentation of the correct matching stimulus, ensuring that monkeys had to make a behavioral response on every trial. The monkeys were rewarded with apple juice for making correct responses and were rewarded randomly at 0% coherence where the sample contained no task-relevant information. During each session, the monkeys performed the task with a set of four familiar stimuli, with which they had many weeks of practice, as well as with a set of four novel stimuli that they had never seen before. Matches occurred on 50% of trials; the other 50% were non-matches selected randomly from the remaining stimuli.Owing to the normalization procedure, familiar and novel stimuli did not differ in terms of low-level characteristics of spatial frequency content and image intensity. Familiar stimuli from four categories were used , and one of the four novel stimuli also came from each of these categories. Fixation was monitored with a scleral search coil and sampled at 200Hz , and the monkeys were required to maintain fixation within a ±1.25° window at all times during the trial. The monkeys completed at least ten trials per condition during each session.t-test, p = 0.87), but significantly lower during recording sessions for familiar stimuli , likely owing to nonspecific factors such as additional wait periods during these sessions.Recordings were made from V4 using standard electrophysiological techniques. We employed a grid system  with eight tungsten microelectrodes . Preoperative magnetic resonance imaging (MRI) was used to identify the stereotaxic coordinates of V4, which was then covered by a recording chamber. To ensure an unbiased estimate of neural activity, we made no attempt to select neurons based on task selectivity. Instead, we advanced each electrode until the activity of one or more neurons was well isolated and then began collecting data. Comparison of the monkeys' performance during the last six training sessions to performance during recording sessions revealed that performance was unchanged for novel objects . The thresholded FPMs shown in To determine whether there were any learning-related changes in the monkeys' fixational eye movements, we performed separate behavioral experiments in which we allowed the monkeys to freely view the sample stimulus for a period of about 2 s. As before, we presented four familiar and four novel stimuli during each session, but we only used two coherence levels of 100% and 45% to allow us to assess whether learning led to changes both for degraded and undegraded stimuli. Monkeys performed around 20 trials for each stimulus at each degradation levels during each session, and we report here the results from a total of six sessions. We identified periods of fixation during free-viewing as periods as periods of at least 100 ms duration during which eye position did not change by more than 0.3°. We then marked off a region of 0.3° × 0.3° around this position and superimposed these regions for all fixations during all relevant trials. By normalizing the volume under this function to a value of 1, we created an FPM for each stimulus. We then applied a single threshold to the FPM for all stimuli and degradation levels. The threshold η was chosen to be an order of magnitude greater than the FPM value corresponding to randomly distributed eye position, i.e., to a value of η = 10 × 1/256t-test evaluated at p < 0.05. Mean firing rates, reported in Neural activity was assessed during a fixed period of 310 ms duration, beginning 50 ms after the onset of the visual stimulus to take response latency into account. Such a period roughly corresponds to the time between saccades during natural viewing conditions. Out of a total population of 116 neurons, 83 task-related neurons were identified as showing significant differences in activity between any of the eight stimuli at any coherence level using a Bonferroni-corrected s) and the neural responses (r):To assess whether learning had any systematic effect on the amount of stimulus-specific information communicated by V4 neurons, we quantified how much information was contained in the pattern of neural firing rates about novel and familiar stimuli separately. This quantity is given by the mutual information between the set of four familiar or novel stimuli and the set of associated firing rates . We thusP(s) is the probability of showing stimulus s, P(r|s) is the probability of observing a response r when stimulus s is presented, and P(r) is the probability of observing response r.where Because calculation of information requires many trials, we computed information for two conditions: degraded and undegraded stimuli. For degraded stimuli, we pooled the coherence levels from 35% to 65%. For undegraded stimuli, we estimated the mutual information for 100% coherence stimuli during the sample period as well as during the probe period on nonmatch trials (to exclude possible movement-related activity). We report here estimates during the probe period because they are based more trials, but results were similar for the sample period. This ensured that information measures for degraded and undegraded stimuli were based in a similar number of trials.fam,100, Ifam,degrad) and novel  stimuli. Note that although across all sessions we employed many more novel than familiar stimuli, each individual neuron from which we recorded during a given session ‘saw' exactly the same number of four familiar and four novel stimuli. We identified highly selective neurons in each population by selecting the 25% neurons that communicated most stimulus information about either novel or familiar stimuli ; i.e., we chose the top 25% of the distribution max. We did this because, owing to our unbiased procedure, our sample contains neurons that did not communicate large amounts of information, and we thus wanted to establish that our conclusions also applied to the neurons that communicated most information. These neurons are shown as white filled circles in n = 62) are shown as gray filled circles. There was significant overlap  between the populations of informative neurons for degraded and undegraded stimuli , indicating that the majority of neurons that were informative for undegraded stimuli were also informative for degraded stimuli. There were no significant differences between informative neurons and the entire population in terms of mean firing rate. Unless otherwise noted, we used paired t-tests to compare information measures obtained for novel and familiar stimuli.For each neuron we estimated four different information values, describing how much stimulus-specific information was contained in its firing rate distributions about undegraded, as well as degraded, familiar (I


The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science “biology.”Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA “genes,” making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This “chemical translation” provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment  on a library of 10 The authors use DNA "genes" to direct the synthesis of combinatorial chemistry libraries, and show in an in vitro selection experiment that specific drugs can be developed Creation of molecular function represents a fundamental challenge. Nature accomplishes the task through evolution, iterating cycles of selection, amplification, and diversification. Multiple generations of selective pressure and reproduction transform a diverse population into one consisting only of molecules fit to survive. Life on this planet thus emerged from a limited chemical palette, comprising proteins, nucleic acids, sugars, lipids, and metabolites. Over the last two decades, technologies that recapitulate this process in the test tube have been developed, and have produced an amazing collection of biopolymers with unprecedented recognition and catalytic properties  libraries represents a first approximation to small-molecule evolution, in that the process roughly mimics the diversification and selection components of evolution. However, amplification and iteration have no functional equivalents in HTS-CC techniques, placing practical limits on library complexity. Amplification and iteration are critical for identifying vanishingly small amounts of material from a complex population. Moreover, these processes make possible the application of bulk selections rather than serial screens to assay libraries, vastly increasing throughput. Accordingly, typical HTS-CC libraries rarely exceed 10 members , whereas members . A state members . If the In order to apply in vitro selection to combinatorial chemistry libraries, each compound must be associated with a gene that specifies its structure. DNA has been utilized previously to record the synthetic history of individual beads in a split-and-pool combinatorial synthesis, but the DNA tags could not direct subsequent resynthesis of the corresponding compound . More reIn vitro selection requires iterated rounds of three steps: conversion of genes to gene products, selection of gene products, and gene amplification . The lasIn general, small-molecule libraries are synthesized by the split-and-pool method which is illustrated in The construct we chose for our DNA support library is shown in Our scheme for DNA-directed split and pool synthesis is shown in We first developed a Sepharose-based resin derivatized with anticodon oligonucleotides complementary to codon sequences . We testWe next addressed chemical synthesis on unprotected DNA. Use of a solid phase in small-molecule synthesis allows for the application of excess reagents, to drive reactions to completion, and simplifies product purification . To realAs an initial chemistry, we chose 9-Fluorenylmethoxycarbonyl [Fmoc]–based peptide synthesis. Synthesis of [Leu]enkephalin on a 340-base ssDNA support, capable of encoding an eight-step synthesis, was analyzed using an electromobility shift assay and the enkephalin-specific 3-E7 antibody . Figure Our chemical translation strategy requires repeated hybridization-directed splitting and coupling of chemical building blocks to DNA. Two different solid phases were utilized for these tasks. To efficiently transfer DNA from anticodon columns to DEAE Sepharose columns, we cyclically pumped 50% dimethylformamide (DMF) over the columns connected in series . ConversTo test and validate our general strategy, we applied in vitro selection to a primarily nonnatural peptide library, with the goal of identifying a high-affinity ligand for the monoclonal antibody 3-E7 . Isolati5-fold by PCR recombination to generate a support library with a complexity of one million, as verified by DNA sequencing  was diversified 10quencing . This liquencing A. The fiIn order to monitor library convergence, DNA from the starting material (round 0) and from after one (round 1) or two (round 2) selection generations was subcloned, and approximately 70 different isolates from each round were sequenced B. In rou1-j2-b3-c4-h5-i6, whereas in the alternate library it was d1-b2-g3-g4-i5-f6. Two rounds of selection enriched the alternate [Leu]enkephalin DNA gene 105-fold (data not shown). The data suggest that little, if any, DNA sequence encoding bias exists in our system. Further, they illustrate the reproducibility of the technology. Together, the results demonstrate conclusively that the DNA display strategy can be used for the in vitro selection of synthetic chemical libraries.To assess generality, we carried out a second [Leu]enkephalin in vitro selection experiment using a peptide library of the same size but constructed with a completely different “genetic code.” Every codon in the alternate library coded for an amino acid different from the one it coded for in the first library. The [Leu]enkephalin codon series in the first library was bPrevious efforts to expand the scope of in vitro selection have utilized nonnatural bases or amino acids incorporated into DNA, RNA, and peptide libraries using polymerases and the ribosome . Such efAn alternative strategy for expanding the chemical diversity of gene products exploits DNA-templated synthesis, where hybridization-induced proximity promotes covalent bond formation . One greDNA display is a general method for the in vitro selection of synthetic combinatorial chemistry libraries. The system is modular, so that chemistry and selection protocols can be easily changed. It can take advantage of existing combinatorial chemistry technology as well as chemical transformations previously carried out in the presence of unprotected DNA . Solid-pIn addition to diverse chemistries, many different library architectures are also possible. The library reported here was synthesized in six encoded steps with ten distinct building blocks per step. However, essentially any combinatorial scheme can be accommodated. The 20-base codon sequences used here were taken from a larger set  of 20-base sequences experimentally verified to exhibit orthogonal hybridization properties .(E) is defined as the geometric increase in the fraction of target molecules in a library that results from a single round of synthesis and selection. Fidelity (F) is defined as the fraction of genes recovered from a completed library synthesis that have been correctly translated. The signal-to-noise characteristic of a selection (S/N) is defined as the ratio of the fraction of target molecules selected to the fraction of nontarget molecules selected. In most cases, the fold enrichment reduces to the simple expression at the right of As a first approximation, the highest possible fold enrichment per round of selection can be determined by considering its relationship to translation fidelity and the signal-to-noise ratio of the selection. Fold enrichment 0f denotes target gene fraction in the selection input and p denotes the probability of a nontarget gene being mistranslated to the target gene product. Biological systems have such high fidelity that F can be considered to equal one. However, the fidelity of chemical translation processes is the product of hybridization specificity and chemistry efficiency raised to the power of the number of steps. It is important to consider these parameters when adapting new chemistries and selections to the DNA display format. S/N of 1000 would give a 10-fold enrichment per round at best. If the library included 1012 unique members, at least 12 rounds would be required to achieve convergence.where 12 distinct small molecules per 30 pmol of DNA starting material, a quantity easily manipulated in a microcentrifuge tube.In addition to influencing convergence rates, fidelity also limits achievable library complexity. The maximum effective library complexity corresponds to the product of Avagadro's number, the moles of library, and the fidelity. Based on our observed 90% hybridization efficiency and 95% chemistry efficiency, extension of the library reported here to 13 synthetic steps would produce 1013.Diversification between rounds of selection by recombination makes possible in vitro evolution of libraries with complexities exceeding the physical library size. Thus, a “best” molecule can be pinpointed without exhaustive testing of all potential species. Starting with a working population of compounds that sparsely sample a chemical space, molecules containing parts of an optimal molecular solution often have a selective advantage relative to siblings, and become enriched. Subsequent recombination processes splice together fragments from the numerous partially optimal molecules to form a globally optimal molecule. Thus, the best structure is found, even if the odds were negligible that it existed in the initial working population. The same principle accounts for the striking success of gene shuffling in protein engineering  and of t6-fold more complex than current synthetic libraries) will test the validity of this belief.DNA display enables the use of genetic tools such as complementation analysis and backcrossing to analyze small-molecule populations. The approach can be used to study molecular evolution without potential biases resulting from experiments restricted to RNA, DNA, and peptide polymers. A general scientific problem that will be directly addressed is the relationship between small-molecule library complexity and the quality of molecules discovered. With biopolymers, more complex libraries yield higher-affinity ligands . HoweverDrug discovery would represent one important application for a small-molecule in vitro selection technology. While the cost of drug discovery has increased continuously over the last decade (from less than $15 billion for research and development in 1996 to more than $25 billion in 2002), the number of new molecular entities approved by the FDA has steadily dropped, from 56 in 1996 to 17 in 2002 . A fast,Future extensions of DNA display include the development of massively parallel array-based splitting strategies for the in vitro selection of low-molecular-weight small-molecule libraries . Massively parallel syntheses will produce compounds that conform better to Lipinski's “rule of five”  and presThe 3-E7 antibody was purchased from Gramsch Laboratories . PANSORBIN cells were purchased from Calbiochem . BSA was purchased from New England Biolabs . Yeast tRNA was purchased from Ambion . The [Leu]enkephalin peptide and all oligonucleotides were purchased from the Stanford PAN Facility .Solid-phase peptide synthesis was carried out as previously described . 5′ aminThe electromobility shift assay was performed as previously described . No plas2, 1X Promega Taq reaction buffer, and 5 U of Taq DNA polymerase . The PCR program was 94 °C for 2.5 min, 58 °C for 1 min, and 72 °C for 15 min. The dsDNA–peptide conjugates were incubated with PANSORBIN cells in 50 μl of Selection Buffer  at 4 °C for 1 h to preclear conjugates that nonspecifically bind to the cells. Then, preclear beads were pelleted by centrifugation and removed. Antibody 3-E7 (0.5 μg) was added to the supernatant and allowed to incubate for 1 h at 4 °C. The solution was then mixed with fresh PANSORBIN cells for 1 h at 4 °C. The cells were pelleted and washed at 25 °C three times with 500 μl of Wash Buffer , followed by a single wash with 500 μl of Selection Buffer. The dsDNA–peptide conjugates were eluted by incubation of the cells with 50 μl of 200 μM [Leu]enkephalin in Selection Buffer for 1 h at 25 °C. Selected genes were amplified from 10 μl of elute supernatant with 25-cycle PCR reactions.ssDNA was converted to double-stranded DNA (dsDNA) by one-cycle PCR with a single end primer. The 50-μl PCR reaction contained 20 μM primer, 200 μM of each dNTP, 5 mM MgClHigh performance liquid chromatography analysis of DNA–peptide conjugates, synthesis of anticodon columns, hybridization and transfer of DNA, library assembly, ssDNA generation, and library isolation were performed as previously described .


Salmonella Enteritidis infections, phage type 4 has been the dominant phage type in most countries in Western Europe during the last years. This is reflected in Salmonella infections among Swedish travellers returning from abroad. However, there are differences in phage type distribution between the countries, and this has also changed over time.Among human Salmonella Enteritidis infections in Swedish travellers from 1997 to 2002, and have compared this with national studies conducted in the countries visited.We used data from the Swedish infectious disease register and the national reference laboratory to describe phage type distribution of S. Enteritidis infections among Swedish travellers returning from some countries in southern Europe was observed, and a previously rare phage type (PT 14b) became one of the most commonly diagnosed that year, continuing into 2002 and 2003.Infections among Swedish travellers correlate well with national studies conducted in the countries visited. In 2001 a change in phage type distribution in Surveillance of infections among returning travellers can be helpful in detecting emerging infections and outbreaks in tourist destinations. The information needs to be communicated rapidly to all affected countries in order to expedite the implementation of appropriate investigations and preventive measures. Salmonella Enteritidis is the most common serovar causing food-borne salmonellosis in humans, causing approximately 80% of salmonellosis cases reported in Europe [S. Enteritidis infections was reported in Europe and North America [S. Enteritidis varies between countries; while phage type (PT) 4 is reported to be dominant in most countries in Western Europe, PT 8 is common in North America and also a few European countries [S. Enteritidis contamination of eggs [S. Enteritidis in recent years [n Europe . During  America -4. The mountries -7. Epideountries ,8. Approountries . Based oountries ,10. Thes of eggs  and are nt years ,12.In Sweden, about 85% of the reported salmonella infections are acquired during travel abroad and the levels in domestic animals and food products is low . TherefoS. Enteritidis infections, describe the phage type distribution of S. Enteritidis isolated from Swedish travellers returning from abroad related to country of infection, and discuss possible reasons for the emergence of a new phage type of S. Enteritidis in 2001. A preliminary analysis of a subset of these data was published as a short letter in the Lancet in 2002 [In this study, we investigate trends in travel related  in 2002 .S.Enteritidis notified to SMI from January 1, 1997 through December 31, 2002.In Sweden, salmonellosis is a mandatory notifiable disease. Both clinicians and laboratories are required to report a case to the infectious disease register at the Swedish Institute for Infectious Disease Control (SMI). Based on the patient's travel history, information on probable country of infection is collected on the notification forms. Diagnosis of salmonella infections is made at regional microbiology laboratories, and all isolates are submitted to the national reference laboratory at SMI for serotyping and phage typing. In this study we have included all cases of To investigate trends in travel-related infections, we collected information on air travel from the Swedish Civil Aviation Administration (CAA). The figFor the geographical description of dominant phage types in the different countries the analysis was restricted to infections acquired in countries in Europe during 1997 to 2002, and to countries from which more than ten cases were reported during this period. A phage type was considered dominant if it represented >30% of the isolates and was at least twice as common as the second most common phage type. If none of the phage types fulfilled these criteria, the two most common phage types that together represented >50% of the cases were defined as the dominant types. The pattern for 1997 to 2000 was compared with the pattern for 2001.S.Enteritidis infections notified during 1997–2002, 11,570 cases (87%) were reported as infected abroad, 1,032 (8%) were reported as infected in Sweden, and information of probable country of infection was not available for 669 (5 %). The total number of cases reported each year varied from 1,598 to 2,629 cases, with the highest being reported in 1999 and the lowest in 2002. Imported cases varied between 1,404 and 2,164. The most common countries of infection during the six-year period were Spain, Greece and Turkey, accounting for 34%, 8% and 4% of all cases, respectively.Of 13,271 cases of S. Enteritidis among Swedish travellers were reported each year during 1997 to 2002. These are all popular destinations for leisure travel among Swedes. Figure For six countries – 'Spain, Greece, Turkey, Poland, Thailand and Portugal – >50 cases of infection with Eighty-six percent  of the isolates from 1997 to 2001 were phage-typed, increasing from 75% in 1997 to 95% in 2001. The most common phage types over the period were PT 4 and PT 1  sent by the Norwegian Public Health Institute gave no response on increases of PT 14b in other countries. Spain reported an outbreak of the same phage in a school in January (unpublished data). But after this event, no further increase was noted. The UK later reported an increased number of the same phage type, both among travellers and among people who had been infected in the UK. However, there the 14b isolates of domestic origin were aerogenic, while isolates associated with travel to Greece were predominantly anaerogenic [The change in phage type distribution observed among Swedish travellers returning from some countries in Southern Europe in 2001 was not observed among inhabitants in the countries visited. In total, the two most common phage types among Swedish travellers were, as in previous years, PT 4 and PT 1. However, the third most common was PT 14b, a phage type hitherto uncommon in Sweden with only 20 to 40 cases reported annually prior to 2001. The majority of the cases were among travellers returning from Greece (90%). More cases were also reported among travellers returning from Spain and Bulgaria than in previous years. During the same time period, an increase in the same phage type had also been registered among Norwegian and Finnish travellers returning from Greece . A requeerogenic . The isoNo explanation for the sudden increase in this phage type among Nordic travellers to different countries in Southern Europe has been found. PT 14b is not a new phage type and outbreaks reported previously have been mainly related to eggs and egg-products  or iSalmonella isolates is not routinely performed in some countries. However, if available, this information will be included in the data reported to Enter-Net. Data from this network has previously been useful in detecting travel-related outbreaks [Our data presented are limited by the small numbers of cases from each country investigated on an annual basis. It is therefore difficult to evaluate trends with any certainty. However, the possibility of using surveillance data of infections among returning travellers to detect emerging pathogens should be further investigated. In addition, data from countries that routinely collect information on travel could be pooled in order to increase the numbers of travel-related infections. In several countries outside Europe, laboratory capacity is limited and it may take a long time to detect the emergence of new pathogens or subtypes. Not all countries in Europe collect information on the probable place of infection and phage typing of utbreaks ,28, and This study demonstrates that surveillance of infections among returning travellers may be helpful in detecting emerging infections and outbreaks in tourist destinations, and provides some useful supplementary data about infectious diseases and trends in other geographical regions. Characterization of isolates from travellers can detect changes in the pathogen and antimicrobial resistance patterns in the destination country. This information may be an important supplement in countries where surveillance systems are deficient or lacking, or where the laboratories have limited capacity to do detailed sub-typing and resistance testing. In addition, infections and outbreaks among tourists may not always affect the local residents and therefore may not be detected by the local public health authorities. If proper investigations, and appropriate prevention and control measures are to be implemented in the countries visited, it is important that the surveillance information compiled from the traveller's home countries is rapidly communicated to the affected countries.None declared.KN performed the data analysis and drafted the manuscript. PJG, YA and BdJ participated in the design and coordination of the study. AO conducted typing and provided advice regarding laboratory issues. JG participated in the design and discussion, and provided advice on data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Xenopus oocyte maturation.The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus, then prepared antibodies specific for five subunits . With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase).We identified cDNA for three α-type subunits  of Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently.Six of α-type subunits of the  Eukaryotic cells, from yeast to human, contain large nonlysosomal proteases called proteasomes . The 26SXenopus and goldfish as α 4 [Xenopus and prepared antibodies for a total of five subunits. Using a set of specific antibodies, we analyzed changes in all α subunits composing the 26S proteasome during the meiotic cell cycle. We demonstrated that 6 of the subunits exist as a heterogeneous population in frog oocytes and identified another subunit in addition to α4 which was modified meiotic cell cycle dependently.Fully grown frog oocytes arrest in the late G2 phase of meiosis. Maturation-inducing hormone (MIH) acts on the oocytes, inducing final maturation and triggering germinal vesicle breakdown (GVBD), and the oocytes arrest again at the second meiotic metaphase until fertilization. The proteasomes are thought to be involved in regulating the maturation and fertilization of oocytes ,7. Previh as α 4 ,9. In thXenopus EST database was conducted using known proteasomal subunit α cDNAs. From the data for each subunit, full-length ORFs were obtained by PCR. The amplified cDNAs were 741, 726 and 786 bp long. The clones encode proteins of 246, 241 and 261 amino acid residues with a predicted molecular mass of 27463, 26402 and 29327 daltons, respectively  [Xenopus 20S proteasome. We named these clones α1_xl, α5_xl and α6_xl  according to a systematic nomenclature [Xenopus 20S proteasome. Overall identity between the subunits was 25.1–38.4 %. A consensus sequence for α-type proteasomal subunits was conserved. Interestingly, a conserved sequence for β-type proteasomal subunits was found in the α3 subunit [A BLAST search of the 2–69.1%) ,13 and y2–69.1%) -16  were raised against purified recombinant proteins. The specificity of the antibodies was examined by immunoblotting with the cytosol fraction and the purified 26S proteasome  ioteasome . The phon et al.  (1996) sn et al. . Recent 96 ioteasn et al. ,29. Cycln et al. . Calcium[Xenopus . It can [Xenopus . PossiblXenopus, there is a possibility that the changes in the spots identified in this study may derive from differential expression of alternative subunits from paralogous genes.Recently, alternative subunits of proteasomes have been identified. In Drosophila where alternative α-type, β-type and 19S cap subunits are expressed from separate genes during spermatogenesis  and in Aα1_xl, α5_xl and α6_xl) of X. laevis were identified.(1) cDNAs for three α-type proteasome subunits (X. laevis.(2) Six subunits but not α7_XL are modified in immature oocytes in X. laevis.(2) α2, α4_XLs are modified during the meiotic cell cycle in Xenopus laevis) were purchased from Jo-hoku Seibutsu Kyozai  and maintained till used. 26S proteasomes were purified from immature oocytes and ovulated oocytes as described [Frogs (escribed .SDS-PAGE was carried out according to the method of Laemmli  (1970). Xenopus EST database was conducted using known proteasomal subunit α cDNAs. From the data obtained for each subunit, independent sequences were linked and the full-length ORF sequences were eliminated. The specific primers for amplification of the full-length ORF were α1: 5'-GGAATTCCATATGTCTCGGGGATCTAGCGCG-3' and 5'-CCGCTCGAGGTCACGCTCAGCTAGTGCAAC-3', α5: 5'-GGAATTCCATATGTTCCTAACCCGCTCCGAG-3' and 5'-CCGCTCGAGGATGTCCTTAATAACTTCCTC-3', and α6: 5'-GGAATTCCATATGTTTCGCAATCAGTATG-3' and 5'-CCGCTCGAGGTGCTCCATAGGCTCCTCCTGC-3'), in which EcoRI (5'end) and XhoI (3'end) recognition sequence was added for cloning to the vector pET21a (Novagen). PCR was carried out using KOD DNA polymerase (TOYOBO) or LA taq DNA polymerase (TaKaRa), with Xenopus ovarian cDNA as a template, and the product was cloned to pET21a. The DNA sequencing was performed using a 377A DNA sequencer (Applied Biosystems). The sequences that include the full-length ORF identified here were deposited into GenBank . Pairwise comparisons of sequence homology were conducted using the Genetyx-Mac ver.12 computer program .Identification and sequence analysis of cDNAs. A BLAST search of the E. coli BL21 (LysE) and purified by SDS-PAGE as described [The recombinant proteins were produced in escribed . Polycloescribed . Anti sebp, base pair; BLAST, basic local alignment search tool; cDNA, DNA complementary to RNA; EST, expressed sequence tags; kDa, kilodalton; NEPHGE, non-equilibrium pH gradient gel electrophoresis; PCR, polymerase chain reaction; SDS-PAGE SDS-polyacrylamide gel electrophoresis; 2D-PAGE, two-dimensional-PAGE.YW carried out cDNA cloning, expression of recombinant proteins, antibody production and 2D-PAGE analysis. MT and RH participated in cDNA cloning, expression of recombinant proteins. YN and KI participated in coordination of the study. TT carried out the protein purification and also participated in the design of the study and drafted the manuscript.