CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery.

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Dietary fatty acids differentially regulate production of TNF-alpha and IL-10 by murine 3T3-L1 adipocytes.

OBJECTIVE: Obesity correlates with increased production of adipocyte-derived cytokines, which may contribute to a chronic subclinical inflammation seen in obese individuals. This study evaluated the ability of specific fatty acids to modulate production of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), and the anti-inflammatory cytokine, interleukin-10 (IL-10), in murine 3T3-L1 adipocytes. Effects on nuclear factor-kappaB (NF-kappaB), a key transcriptional activator of the inflammatory cascade, and suppressor of cytokine signaling 3 (SOCS-3), a negative regulator of cytokine signaling, were also determined. METHODS AND PROCEDURES: Adipocytes were incubated for 24 and 48 h with and without 50 or 500 micromol/l of palmitic acid, oleic acid, or docosahexaenoic acid, (DHA). Effects on gene expression and protein secretion of TNF-alpha and IL-10 were determined using real-time PCR and a murine multipex RIA kit. SOCS-3 expression was determined by northern blotting and NF-kappaB binding activity was assessed using a commercially available assay. RESULTS: Adipocytes treated for 24 h with palmitic acid exhibited a 70% increase in TNF-alpha production and up to a 75% decrease in IL-10 production, relative to untreated cells. In contrast, DHA treatment had no effect on TNF-alpha, but increased IL-10 production twofold. No effect of oleic acid was seen on either TNF-alpha or IL-10 production. Similar results were obtained during a 48-h incubation. Furthermore, NF-kappaB DNA-binding activity increased fourfold in response to palmitic acid and decreased 60% in response to DHA. Expression of SOCS-3 increased twofold in DHA-treated cells. DISCUSSION: In aggregate, these results suggest that dietary fatty acids act directly on adipocytes to modulate cytokine production. As circulating fatty acids levels are chronically elevated in obese individuals, this effect may account in part for obesity-associated inflammation.

Viral mutations enhance the Max binding properties of the vMyc b-HLH-LZ domain.

Interaction with Max via the helix-loop-helix/leucine zipper (HLH-LZ) domain is essential for Myc to function as a transcription factor. Myc is commonly upregulated in tumours, however, its activity can also be potentiated by virally derived mutations. vMyc, derived from the virus, MC29 gag-Myc, differs from its cellular counterpart by five amino acids. The N-terminal mutation stabilizes the protein, however, the significance of the other mutations is not known. We now show that vMyc can sustain longer deletions in the LZ domain than cMyc before complete loss in transforming activity, implicating the viral mutations in contributing to Myc:Max complex formation. We confirmed this both in vitro and in vivo, with loss of Max binding correlating with a loss in the biological activity of Myc. A specific viral mutation, isoleucine383>leucine (I383>L) in helix 2 of the HLH domain, extends the LZ domain from four to five heptad repeats. Significantly, introduction of I383>L into a Myc mutant that is defective for Max binding substantially restored its ability to complex with Max in vitro and in vivo. We therefore propose that this virally derived mutation is functional by significantly contributing to establishing a more hydrophobic interface between the LZs of Myc and Max.

Serum high molecular weight complex of adiponectin correlates better with glucose tolerance than total serum adiponectin in Indo-Asian males.

AIMS/HYPOTHESIS: It is well established that total systemic adiponectin is reduced in type 2 diabetic subjects. To date most studies have been concerned with the singular full-length protein or proteolytically cleaved globular domain. It is, however, apparent that the native protein circulates in serum as a lower molecular weight hexamer and as larger multimeric structures of high molecular weight (HMW). In this study we address the clinical significance of each form of the protein with respect to glucose tolerance. METHODS: Serum was obtained from 34 Indo-Asian male subjects (BMI 26.5+/-3.1; age 52.15+/-10.14 years) who had undertaken a 2-h oral glucose tolerance test. An aliquot of serum was fractionated using velocity sedimentation followed by reducing SDS-PAGE. Western blots were probed for adiponectin, and HMW adiponectin as a percentage of total adiponectin (percentage of higher molecular weight adiponectin [S(A)] index) was calculated from densitometry readings. Total adiponectin was measured using ELISA; leptin, insulin and IL-6 were determined using ELISA. RESULTS: Analysis of the cohort demonstrated that total adiponectin (r = 0.625, p = 0.0001), fasting insulin (r = -0.354, p = 0.040) and age (r = 0.567, p = 0.0001) correlated with S(A). S(A) showed a tighter, inverse correlation with 2-h glucose levels (r = -0.58, p = 0.0003) than total adiponectin (r = -0.38, p = 0.0001). CONCLUSIONS/INTERPRETATION: This study demonstrates the importance of the S(A) index as a better determinant of glucose intolerance than measurements of total adiponectin. Our findings suggest that HMW adiponectin is the active form of the protein.

Safe introduction of warfarin for thrombotic prophylaxis in atrial fibrillation requiring only a weekly INR.

A new protocol for initiating warfarin therapy was introduced to reduce the workload in the Anticoagulant Clinic. A total of 200 outpatients, with a median age of 74 years, requiring anticoagulation for atrial fibrillation, commenced warfarin 3 mg daily for 1 week. Patients were initially seen weekly, and subsequent warfarin doses were dictated by the International Normalized Ratio (INR) on days 8 and 15; 84% of patients followed the protocol correctly: of these 86% had an INR > or =2 by day 15 and >98% had INR >2 by day 22. By day 22, 58% of patients achieved a stable dose, 85% by day 29 and >95% by day 36. Day 8 INR was predictive of the final maintenance dose required. No patient suffered any thrombotic or haemorrhagic complications in the first month: only three patients had an INR >3 on day 8, and 11 patients had an INR >4 on day 15. Patient age and sex were not sufficiently related to warfarin requirement to provide useful predictive information. This protocol, requiring only weekly INRs, has proved safe and effective for outpatient warfarinization, and has reduced clinic attendances in this population.

Differences in adiponectin protein expression: effect of fat depots and type 2 diabetic status.

Adiponectin is an adipocyte-derived hormone associated with insulin sensitivity and atherosclerotic risk. As central rather than gluteofemoral fat is known to increase the risk of type 2 diabetes and cardiovascular disease, we investigated the mRNA and protein expression of adiponectin in human adipose tissue depots. RNA was extracted from 46 human adipose tissue samples from non-diabetic subjects aged 44.33 +/- 12.4 with a BMI of 28.3 +/- 6.0 (mean +/- SD). The samples were as follows: 21 abdominal subcutaneous, 13 omentum, 6 thigh; samples were also taken from diabetic subjects aged 66.6 +/- 7.5 with BMI 28.9 +/- 3.17; samples were: 6 abdominal subcutaneous; 3 thigh. Quantitative PCR and Western analysis was used to determine adiponectin content. Protein content studies determined that when compared with non-diabetic abdominal subcutaneous adipose tissue (Abd Sc AT) (values expressed as percentage relative to Abd Sc AT -100 %). Adiponectin protein content was significantly lower in non-diabetic omental AT (25 +/- 1.6 %; p < 0.0001, n = 6) and in Abd Sc AT from diabetic subjects (36 +/- 1.5 %; p < 0.0001, n = 4). In contrast, gluteal fat maintained high adiponectin protein content from non-diabetic patients compared with diabetic patients. An increase in BMI was associated with lower adiponectin protein content in obese ND Abd Sc AT (25 +/- 0.4 %; p < 0.0001). These findings were in agreement with the mRNA expression data. In summary, this study indicates that adiponectin protein content in non-diabetic subjects remains high in abdominal subcutaneous fat, including gluteal fat, explaining the high serum adiponectin levels in these subjects. Omental fat, however, expresses little adiponectin. Furthermore, abdominal and gluteal subcutaneous fat appears to express significantly less adiponectin once diabetic status is reached. In conclusion, the adipose tissue depot-specific expression of adiponectin may influence the pattern of serum adiponectin concentrations and subsequent disease risk.

[Influence of planting density and precipitation on N2O emission from a winter wheat field].

To investigate the impact of plant density on N2O emission from winter wheat field and the cause of seasonal variation in the emission, field experiment with four planting rates of 0, 90, 180 and 270 kg/ha was conducted at the Jiangning County near Nanjing during 1999-2000 winter wheat growing season. Data of the field measurements indicated that the N2O emission rates during the season from planting to overwintering were not influenced by the plant density, while the emission was positively correlated with the planting density during the season from turning green to maturity. The emissions from the field plots with planting rates of 0 and 90 kg/ha were not found to be significantly different. A further analysis suggested that the seasonal variation of N2O emission be mainly influenced by precipitation, which could be quantitatively described by an exponential function of a weighted average precipitation of 6-day period before measurement.

Absence of congenital prethrombotic disorders in children with Legg-Perthes disease.

Resistance to activated protein C (RPCA) and other congenital prethrombotic disorders have been recently reported to be strongly associated with Legg-Perthes disease. RPCA and deficiencies of protein C, protein S, and antithrombin III were sought in 22 children with Legg-Perthes disease. Detection of the factor V Leiden mutation was found in children with RPCA. Twenty-two healthy children paired by age and sex served as controls. The prevalence of congenital prethrombotic disorders was not found to differ significantly among patients with Legg-Perthes disease and among control subjects. Only one patient had RPCA; this patient was heterozygous for the factor V Leiden mutation. Twenty patients and all the control subjects had entirely normal coagulation results. The authors conclude that unless more data become available, RPCA and deficiencies of protein C, protein S, and antithrombin III should not be considered associated with Legg-Perthes disease.

Improving the efficiency of survey research in postoperative patients.

STUDY OBJECTIVE: To increase the contact rate with eligible patients for quality assurance/improvement surveys by modifying survey rounds to accommodate the schedules of individual nursing units. DESIGN: Two-phase, interventional time series study. SETTING: Postoperative inpatients at a university hospital. PATIENTS: 498 adult postoperative inpatients who remained hospitalized during the second postoperative day. INTERVENTIONS: Between the first and second measurement periods, efforts were made to learn the schedule of each nursing unit and to improve the efficiency of survey rounds so that a larger proportion of patients could be contacted. MEASUREMENTS AND MAIN RESULTS: The contact rate for eligible patients was improved from 66% to 80% (p < 0.01). Improvement during the second period was attributed to fewer patients being away from the nursing unit (20% vs. 12%, p < 0.05) or otherwise occupied by attending physicians on rounds (9% vs. 4%, p < 0.05). CONCLUSION: Strategies individualized to patient care units can improve the efficiency and credibility of inpatient survey research. We describe the strategies most helpful in improving the efficiency of survey rounds at one medical center.

[Cerebral vascular complication of hyperhomocysteinemia. Controlling thromboembolic complications with folates]. / Accident vasculaire cérébral avec hyperhomocystéinémie. Arrêt des accidents thromboemboliques sous folates.

BACKGROUND: Young patients who experience cardiovascular events may have raised levels of homocysteine. There may be several causes for this hyperhomocysteinemia. CASE REPORT: Cerebrovascular disease occurred in a 40-year-old female smoker with hyperhomocysteinemia. This patient subsequently had several episodes of thromboembolism involving the brain and lower limb arteries. Prothrombin concentration was difficult to control with antivitamin K anticoagulants. Investigations to identify a genetic cause of hyperhomocysteinemia revealed that she was homozygous for the C677T mutation on the methylenetetrahydrofolate reductase gene. There was no G1691A mutation of the factor V gene, a risk factor for familial thrombosis. Supplementation with folic acid successfully halted episodes of thromboembolism (follow-up 2 years) and prothrombin levels stabilized under treatment. DISCUSSION: The C677T mutation, which is common in the general population (15.7%), cannot explain the effect of folate supplementation alone. Other mutations affecting homocysteine metabolism could have a potentializing effect on vascular events.

Comparison of acceleration subjects to other populations: spinal anomaly distribution.

Two Armstrong Laboratory (AL) human volunteer subject panels (sustained and impact acceleration) at Wright Patterson AFB, OH, were compared to each other and to other samples of different populations in terms of spinal anomalies. These sample populations were obtained from the scientific literature: French, Norwegian, Netherlands, and U.S. pilots; U.S. Air Force (AF) and Navy subjects, and from representative "normal" civilian populations, and then compared using the proportion parameters for various spinal anomalies. There were only a few common parameters between the two panels and between each panel compared with the foreign military, human subjects, and "normal" population. However, there were two to six times as many similar spinal anomaly incidence rates between the AL panels and the U.S. pilot sample. It was reassuring that the AL subject panels used in AF acceleration research have more in common with AF pilots than other populations in regards to spinal anomalies, even though the pilot sample may not be representative of the true pilot population. Recommendations are to establish a common reference point in nomenclature and description of spinal anomalies (modeled after the French) and to start collecting spinal radiographs on all U.S. pilots. These radiographs would not be for screening but for establishing a database following the occupational pathology of flying. This data would also facilitate comparisons with research acceleration panels, as well as with foreign air forces. Informed decisions can then be made regarding screening criteria for the future as aircraft and ejection seat performance envelopes continue to expand.

Data security and patient confidentiality: the manager's role.

The maintenance of patient confidentiality is of utmost importance in the doctor patient relationship. With the advent of networks such as the National Health Service Wide Area Network in the UK, the potential to transmit identifiable clinical data will become greater. Links between general practitioners (GPs) and hospitals will allow the rapid transmission of data which if intercepted could be potentially embarrassing to the patient concerned. In 1994 the British Medical Association launched a draft bill on privacy and confidentiality and in association with this bill it is pushing for encryption of all clinical data across electronic networks. The manager's role within an acute hospital, community units and general practice, is to ensure that all employees are aware of the principles of data protection, security of hospital computer systems and that no obvious breaches of security can occur at publicly accessible terminals. Managers must be kept up to date with the latest developments in computer security such as digital signatures and be prepared to instigate these developments where practically possible. Managers must also take responsibility for the monitoring of access to terminals and be prepared to deal severely with staff who breach the code of confidentiality. Each manager must be kept informed of employees status with regard to their 'need to know' clearance level and also to promote confidentiality of patient details throughout the hospital. All of the management team must be prepared to train new staff in the principles of data security as they join the organisation and recognise their accountability if the programme fails. Data security and patient confidentiality is a broad responsibility in any healthcare organisation, with the Chief Executive accountable. In family practice, the partners are responsible and accountable. The British Medical Association believes as a matter of policy, that allowing access to personal health data without the patients consent, except in a legally allowable situation, should be a statutory offence.

The surgical management of carotid artery stenosis in patients with previous neck irradiation.

BACKGROUND: A history of therapeutic irradiation to the neck complicates the management of carotid artery occlusive disease. Serious surgical concerns are raised regarding alternative incisions, difficult dissections, and adequate wound closure. Pathology may be typical atherosclerotic occlusive disease or radiation-induced arteritis. In order to establish guidelines for the treatment of these patients, we have reviewed our operative experience. PATIENTS AND METHODS: A review of our operative experience over the past 15 years revealed 10 patients with a history of prior irradiation to the neck who underwent 14 carotid operations. RESULTS: The indications for radiation included laryngeal carcinoma and lymphoma. Five patients had undergone previous radical neck dissections, and four patients had permanent tracheostomies. The surgical indications were asymptomatic high-grade stenosis in 7 cases, transient ischemic attack in 4 cases, stroke in 2 cases, and a pseudoaneurysm in 1 case. Conventional carotid endarterectomy with patch angioplasty was used in 10 of the 14 operations. In the remaining four operations, saphenous vein interposition grafting was utilized to replace the diseased segment of carotid artery secondary to a panarteritis. Wound closure required dermal grafting in two of five cases where surgery was performed ipsilateral to a prior radical neck dissection. One perioperative cerebral infarction occurred; there were no other neurologic or non-neurologic complications. All patients are doing well in one- to five-year follow-up, with serial postoperative duplex scans demonstrating no signs of recurrent stenosis. CONCLUSIONS: Patients with a history of irradiation to the neck should be screened for the presence of carotid disease. Carotid occlusive disease should be treated surgically in these patients with the usual indications. Intraoperative surgical management is similar to that of non-irradiated patients. Concerns about difficulty in achieving an adequate endarterectomy plane and about problems with wound closure have generally been unfounded.

Detection of tube agglutination 37 degrees C-only antibodies by solid-phase red cell adherence.

There are no published data on the detection of tube agglutination (TA) 37 degrees C-only antibodies by solid-phase (SP) red cell adherence assays using anti-IgG-coated indicator red cells. Thirteen examples of TA 37 degrees C-only antibodies were tested by conventional SP methods. Four TA 37 degrees C-only antibodies failed to react by SP. Three were anti- Lea, considered clinically insignificant, and one was anti-E, an antibody of potential clinical significance. The remaining nine TA 37 degrees C-only antibodies reacted by SP, including three anti-c, two anti- D, two anti-E, one anti-N, and one anti-M. The anti-M reacted with indicator red cells that lacked the red cell antigen and failed to react with IgG-coated indicator red cells whose anti-IgG component had been neutralized, indicating the antibody contained an IgG component. Two anti-D and one anti-c continued to react in an SP test using neutralized anti-IgG antigen-positive indicator red cells, i.e., indicator binding independent of antiglobulin, suggesting an IgM nature to these antibodies. Therefore, many TA 37 degrees C-only antibodies can be detected by SP either through detection of an IgG component by the anti-IgG of the indicator red cells, or through IgM crosslinking of antigen-positive indicator red cells to antigen-positive SP reagent red cell membranes.

Tunnel restorations: a long-term pilot study over a minimum of five years.

OBJECTIVES: The purpose of this study was to investigate the long-term performance of tunnel restorations. METHODS: Thirty-three glass polyalkenoate (ionomer) or cermet restorations were placed in the premolar or first molar teeth of 25 patients. Conservative amalgam control restorations were the means of treatment in 14 similar teeth of the same group. Review as to performance consisted of a regular clinical and radiographic examination and the criteria for failure were recurrent caries, fracture of the marginal ridge or gross loss of restorative material. RESULTS: After 3 years all types of restoration were satisfactory. By approximately 5 years one-quarter of the glass ionomer and 10% of the cermet restorations had failed. All the failures after any period of service could be replaced by conservative amalgam fillings. None of the controls required replacement. CONCLUSION: While tunnel restorations employing glass ionomer material and the cermet variant do enjoy a degree of success, this preliminary work suggests that small restorations of dental amalgam are more successful in the long term.

Cognitive task classification based upon topographic EEG data.

EEG from 19 electrodes was used to classify which of 14 tasks each of seven subjects had performed. Stepwise discriminant analysis (SWDA) was used to classify the tasks based upon training on one half of the spectrally analyzed 1 min of data. Eighty six percent correct classification was achieved using principle components analysis (PCA) to determine the EEG bands to be used by the SWDA. Other approaches to deriving the EEG bands met with lower levels of success. The results indicate that frequency and topographical information about the EEG provides useful knowledge with regard to the nature of cognitive activity. Higher frequencies provided much of the information used by the classifier. The utility of this approach is discussed with regard to evaluating operator state in the work environment.