Dissociative symptoms among community health service users in Hong Kong: a longitudinal study of clinical course and consequences.

Background: Previous studies have demonstrated the high prevalence of dissociative symptoms and their association with considerable healthcare costs. However, there is a lack of studies that describe whether dissociative symptoms persist and lead to other clinical outcomes over time in the community.Objectives: This study investigated the persistence, predictors, and consequences of dissociative symptoms in the community.Methods: We analyzed longitudinal data in a sample of community health service users in Hong Kong (N = 173).Results: A relatively high proportion (63.6%) of participants with baseline dissociative symptoms continued to exhibit dissociative symptoms after approximately 9 months. Baseline non-betrayal trauma predicted subsequent dissociative symptoms (ß = .141, p = .024). Participants with baseline dissociative symptoms were more likely to have received subsequent emergency mental health services (9.1% vs 0.7%, p = .005). Baseline dissociative symptoms significantly predicted subsequent post-traumatic symptoms (ß = .165 to .191, p < .05) and difficulty in social and occupational participation (ß = -.152 to -.182, p < .05) even after controlling for baseline scores, trauma exposure, and use of professional support. The predictive role of dissociative symptoms on subsequent disturbances in self-organization symptoms and social participation difficulty remained significant after applying the Bonferroni correction.Conclusions: This is one of the very few studies showing that dissociative symptoms are persistent to a certain degree and could predict other symptoms and subsequent impairments even in community settings. Factors that affect the trajectory of dissociative symptoms should be further investigated. Regular screening for dissociative symptoms is recommended. Considering its prevalence, persistence, and clinical and social consequences, dissociation should be given greater public health attention.

Dissociative symptoms have been linked to considerable healthcare costs.The persistence and consequences of dissociation in the community had not been previously reported.This study showed that dissociative symptoms persisted to a certain degree and predicted subsequent impairments after approximately 9 months.Dissociation should be given greater public health attention.

Efficacy of beetroot juice on reducing blood pressure in hypertensive adults with autosomal dominant polycystic kidney disease (BEET-PKD): study protocol for a double-blind, randomised, placebo-controlled trial.

BACKGROUND: In autosomal dominant polycystic kidney disease (ADPKD) impaired nitric oxide (NO) synthesis, in part, contributes to early-onset hypertension. Beetroot juice (BRJ) reduces blood pressure (BP) by increasing NO-mediated vasodilation. The aim of this double-blind, randomised, placebo-controlled study is to test the hypothesis that BRJ reduces systolic and diastolic clinic BP in hypertensive adults with ADPKD. METHODS: Participants with ADPKD and treated hypertension (n = 60) will be randomly allocated (1:1) to receive a daily dose of either nitrate-replete (400 mg nitrate/day) or nitrate-deplete BRJ for 4 weeks. The co-primary outcomes are change in mean systolic and diastolic clinic BP before and after 4 weeks of treatment with daily BRJ. Secondary outcomes are changes in daily home BP, urinary albumin to creatinine ratio, serum and salivary nitrate/nitrite levels and serum asymmetric dimethylarginine levels before and after 4 weeks of BRJ. DISCUSSION: The effect of BRJ in ADPKD has not been previously tested. BRJ is an accessible, natural dietary supplement that, if effective, will provide a novel adjunctive approach for treating hypertension in ADPKD. TRIAL REGISTRATION: ClinicalTrials.gov NCT05401409. Retrospectively registered on 27th May 2022.

Is it safe to take Radix Salvia Miltiorrhiza - Radix Pueraria Lobate product with warfarin and aspirin? A pilot study in healthy human subjects.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Salvia Miltiorrhiza (Danshen) and Radix Pueraria Lobate (Gegen) are officially listed in the Chinese Pharmacopoeia and have long been used together as a Compound Chinese Traditional Medicine (CCTM) for treatment of coronary heart diseases, which are often co-administered with aspirin or warfarin to patients suffering from cardiovascular diseases. AIM OF STUDY: Since significant pharmacokinetic and pharmacodynamic interactions between Danshen-Gegen (DG) formula and aspirin/warfarin have been observed in our previous rat studies, the current study was proposed aiming to further verify such pharmacokinetic and pharmacodynamic interactions in healthy human subjects and explore related mechanisms. MATERIALS AND METHODS: A 5-day, multiple dose, five-session clinical trial has been carried out (n = 14) with 2-week washout periods between sessions, during which the subjects would receive different combinations of the medications. Plasma samples were collected for pharmacokinetic evaluation, and whole blood samples were collected for pharmacodynamic evaluation. In addition, an in-vitro mechanistic study is conducted to investigate the role of danshensu on the anti-thrombotic and anti-platelet aggregation effects of warfarin and aspirin respectively. RESULTS: Significant pharmacokinetic and pharmacodynamic herb-drug interactions were observed in healthy human subjects. pharmacokinetically, co-administration of DG with aspirin or warfarin could lead to a moderately increased AUC0→t of aspirin and a decreased AUC0→t of 7-hydroxyl warfarin respectively. The systemic exposure of danshensu (DSS, the marker component of DG) would be significantly increased after co-administration with warfarin. Pharmacodynamically, a reduction in systemic thromboxane B2 concentration was noticed after administration of DG with aspirin, which could be associated with the increased systemic exposure of aspirin and the synergistic effect of danshensu, aspirin and salicylic acid on cyclooxygenase (COX) inhibition. An offset on the warfarin induced soluble thrombomodulin induction was observed after its co-administration with DG, which could be partially attributed to the COX-2 inhibition effect of danshensu. CONCLUSION: Our results indicated that co-administration of DG with aspirin/warfarin would lead to significant pharmacokinetic and pharmacodynamic herb-drug interactions in healthy human subjects.

Two-Port Pars Plana Anterior and Central Core Vitrectomy (Lam Floaterectomy) in Combination With Phacoemulsification and Intraocular Lens Implantation Under Topical Anesthesia for Patients with Cataract and Significant Floaters: Results of the First 50 Consecutive Cases.

PURPOSE: To study the safety and efficacy of 2-port pars plana anterior and central core vitrectomy (Lam floaterectomy) in combination with phacoemulsification (phaco) and intraocular lens implantation (IOL) for patients with cataract and significant floaters under topical anesthesia. DESIGN: Retrospective review of the first 50 consecutive cases. METHODS: A standardized treatment protocol was used for patients with cataract and significant (moderate to severe) floaters (duration > 3 months). Data analysis included intraoperative and postoperative complications, floater status, and patient satisfaction. RESULTS: There were 50 eyes (38 patients) with a male-to-female ratio of 1 to 2.3. Twelve patients had bilateral eye surgeries. Mean age was 58.10 ± 9.85 years (range, 39-83). All patients completed the 3-month follow-up. One eye had mild vitreous hemorrhage at the end of surgery arising from sclerotomy wound oozing. No other intraoperative compli-cations were encountered. Postoperatively, there was 1 case of transient hypotony and 1 case of congestion at sclerotomy wound. No cases of retinal break or detachment, or clinically significant macular edema, were reported. There were 5 cases (10%) of mild residual floaters and 1 case (2%) of floater recurrence. Total floater clearance rate was 88%. Patient satisfaction rates were 80%, 14%, 6%, and 0% for very satisfied, satis-fied, acceptable, and unsatisfied, respectively. CONCLUSIONS: The 3-month results in terms of safety and efficacy of the Lam floaterectomy in combination with phaco and IOLfor patients with cataract and significant floaters under topical anesthesia are encouraging. Further larger-scale, prospective, multicenter studies seem warranted.

Identifying and integrating consumer perspectives in clinical practice guidelines on autosomal-dominant polycystic kidney disease.

AIM: This study aimed to identify consumer perspectives on topics and outcomes to integrate in the Kidney Health Australia Caring for Australasians with Renal Impairment (KHA-CARI) clinical practice guidelines on autosomal-dominant polycystic kidney disease (ADPKD). METHODS: A workshop involving three concurrent focus groups with 18 consumers (patients with ADPKD (n = 15), caregivers (n = 3)) was convened. Guideline topics, interventions and outcomes were identified, and integrated into guideline development. Thematic analysis was used to analyse the reasons for their choices. RESULTS: Twenty-two priority topics were identified, with most focussed on non-pharmacological management (diet, fluid intake, physical activity, complementary medicine), pain management and psychosocial care (mental health, counselling, cognitive and behavioural training, education, support groups). They also identified 26 outcomes including quality of life (QoL), progression of kidney disease, kidney function, cyst growth and nephrotoxity. Almost all topics and outcomes suggested were identified by health professionals with the exception of five topics/outcomes. Six themes reflected reasons for their choices: clarifying ambiguities, resolving debilitating pain, concern for family, preparedness for the future, taking control and significance of impact. CONCLUSION: Although there was considerable concordance between the priority topics and outcomes of health professionals and consumers for guidelines of ADPKD, there was also important discordance with consumers focused on fewer issues, but particularly on lifestyle, psychosocial support, pain, and QoL and renal outcomes. Active consumer engagement in guidelines development can help to ensure the inclusion of patient-centred recommendations, which may lead to better management of disease progression, symptoms, complications, and psychosocial impact.

Bench to bed evidences for pharmacokinetic and pharmacodynamic interactions involving oseltamivir and chinese medicine.

Oseltamivir (OA), an ethyl ester prodrug of oseltamivir carboxylate (OC), is clinically used as a potent and selective inhibitor of neuraminidase. Chinese medicines have been advocated to combine with conventional drug for avian influenza. The current study aims to investigate the potential pharmacokinetic and pharmacodynamic interactions of a Chinese medicine formula, namely, Yin Qiao San and Sang Ju Yin (CMF1), commonly used for anti-influenza in combination with OA in both rat and human, and to reveal the underlined mechanisms. It was found that although C max, AUC and urinary recovery of OC, as well as metabolic ratio (AUCOC/AUCOA), were significantly decreased in a dose-dependent manner following combination use of CMF1 and OA in rat studies (P < 0.01), such coadministration in 14 healthy volunteers only resulted in a trend of minor decrease in the related parameters. Further mechanistic studies found that although CMF1 could reduce absorption and metabolism of OA, it appears to enhance viral inhibition of OA (P < 0.01). In summary, although there was potential interaction between OA and CMF1 found in rat studies, its clinical impact was expected to be minimal. The coadministration of OA and CMF1 at the clinical recommended dosages is, therefore, considered to be safe.

Anodal transcranial direct current stimulation increases brain intracellular pH and modulates bioenergetics.

Transcranial direct current stimulation is an emerging treatment for brain disorders but its mode of action is not well understood. We applied 10 min 1 mA anodal transcranial direct current stimulation (tDCS) inside the bore of a 3 T MRI scanner to the left dorsolateral prefrontal cortex of 13 healthy volunteers (aged 19-28 yr) in a blinded, sham-controlled, cross-over design. Brain bioenergetics were measured from the left temporo-frontal region using 31P magnetic resonance spectroscopy before, during and for 20 min following tDCS. Brain pH rose during tDCS and remained elevated afterwards. Phosphomonoesters were significantly decreased while inorganic phosphate (Pi) also fell. Partial-least squares discriminant analysis of the data revealed two significantly different subject groups: one where phosphocreatine (PCr), ATP and Pi fell along with a larger increase in pH and one where PCr and ATP increased along with a smaller increase in pH and a slower and more sustained decrease in Pi. Group membership was predicted by baseline pH and ATP. We interpreted the effects of tDCS as driving two biochemical processes: cellular consumption of ATP causing hydrolysis of PCr via the creatine kinase reaction driving the increase in pH; synthesis of ATP and PCr by mitochondria with concomitant drop in Pi and phosphomonoester levels.

Establishing the pharmaceutical quality of Chinese herbal medicine: a provisional BCS classification.

The Biopharmaceutical Classification System (BCS), which is a scientific approach to categorize active drug ingredient based on its solubility and intestinal permeability into one of the four classes, has been used to set the pharmaceutical quality standards for drug products in western society. However, it has received little attention in the area of Chinese herbal medicine (CHM). This is likely, in part, due to the presence of multiple active components as well as lack of standardization of CHM. In this report, we apply BCS classification to CHMs provisionally as a basis for establishing improved in vitro quality standards. Based on a top-200 drugs selling list in China, a total of 31 CHM products comprising 50 official active marker compounds (AMCs) were provisionally classified according to BCS. Information on AMC content and doses of these CHM products were retrieved from the Chinese Pharmacopoeia. BCS parameters including solubility and permeability of the AMCs were predicted in silico (ACD/Laboratories). A BCS classification of CHMs according to biopharmaceutical properties of their AMCs is demonstrated to be feasible in the current study and can be used to provide a minimum set of quality standards. Our provisional results showed that 44% of the included AMCs were classified as Class III (high solubility, low permeability), followed by Class II (26%), Class I (18%), and Class IV (12%). A similar trend was observed when CHMs were classified in accordance with the BCS class of AMCs. Most (45%) of the included CHMs were classified as Class III, followed by Class II (16%), Class I (10%), and Class IV (6%); whereas 23% of the CHMs were of mixed class due to the presence of multiple individual AMCs with different BCS classifications. Moreover, about 60% of the AMCs were classified as high-solubility compounds (Class I and Class III), suggesting an important role for an in vitro dissolution test in setting quality control standards ensuring consistent biopharmaceutical quality for the commercially available CHM products. That is, provisionally, more than half of the AMCs of the top-selling CHMs included in this study would be candidates for a bioequivalence (BE) biowaiver, based on WHO recommendations and EMEA guidelines. Thus a dissolution requirement on these AMCs would represent a significant advance in the pharmaceutical quality of CHM today.

Quantum dots exhibit less bioaccumulation than free cadmium and selenium in the earthworm Eisenia andrei.

The present study addresses the bioaccumulation behavior of cadmium selenide quantum dots by Eisenia andrei earthworms in a terrestrial environment. Earthworms were exposed to quantum dot-treated soil for up to 4 wk and analyzed for cadmium and selenium concentration using inductively coupled plasma mass spectrometry. Results were compared with those from earthworms exposed to cadmium nitrate and selenious acid, as positive controls, and those exposed in untreated soil (negative control). Earthworms exposed to quantum dots showed significant bioaccumulation of cadmium and selenium (5.3- and 1.5-fold higher concentration over negative controls, respectively) after 4 wk. Over the same 4 wk, positive control earthworms accumulated 9.2- and 2.2-fold higher cadmium and selenium, respectively, than negative controls for a much more substantial final body burden of the 2 elements. The concentrations also increased with exposure time; cadmium concentrations increased from 3600 ± 310 ng/g to 8080 ± 660 ng/g, from 1 to 4 wk, suggesting that further bioaccumulation may take place with even longer exposure time. The molar ratio of cadmium to selenium in the quantum dot-exposed worms (6.2) is closer to the ratios seen in positive control worms (7.2) than to the pure quantum dots (1.8), which implies that quantum dots are taken up predominantly in the degraded form. The results suggest that chemical modification of quantum dots to protect them from environmental degradation could potentially reduce bioaccumulation of the nanoparticles by earthworms.

Randomized double-masked controlled trial comparing pain scores with and without the use of supplementary 2% lidocaine gel in LASIK.

PURPOSE: To compare pain scores with and without supplementary topical 2% lidocaine gel in patients undergoing simultaneous bilateral laser-assisted in situ keratomileusis (LASIK) under topical anesthesia using 0.5% proparacaine eye drops. DESIGN: Randomized double-masked placebo-controlled trial. METHODS: Fifty-one Chinese subjects (102 eyes, with 51 eyes in each arm) were included. One eye was randomly allocated to have supplementary 2% lidocaine gel while the other eye received carbomer gel as control, in addition to topical 0.5% proparacaine. The pain scores for each eye during microkeratome flap creation, during laser ablation, and at 15, 30, and 45 minutes after LASIK were assessed. An overall pain score of the LASIK procedure was also obtained. Primary outcome measures were pain scores during and after LASIK. Secondary outcomes included need for additional topical anesthesia, patient cooperation score, and duration and complications of surgery. RESULTS: In the 2% lidocaine gel-treated group, the pain scores were significantly lower during microkeratome flap creation and laser ablation, and postoperatively at 30 and 45 minutes (P<.05 for all). Patients in the lidocaine gel group required less additional topical anesthesia (P=.0004) and were more cooperative (P=.019) as compared to the carbomer gel group. No surgical or postoperative complications were observed. CONCLUSIONS: The use of supplementary 2% lidocaine gel in LASIK is effective in lowering the pain experienced during and up to 45 minutes after LASIK.

A synopsis of current haemophilia care in Hong Kong.

OBJECTIVE: To provide a synopsis of current haemophilia care in Hong Kong. DESIGN: Retrospective survey. SETTING: All haematology units of the Hospital Authority in Hong Kong. PATIENTS: All patients with haemophilia A and haemophilia B. RESULTS: To date, there were 222 mild-to-severe haemophilia patients (192 type A, 30 type B) under regular public care in Hong Kong (43% were considered severe, 33% moderate, and 24% mild), which gave a crude prevalence of 6.8/100 000 male inhabitants. A total of 12.8 million units of Factor VIII and 3 million units of Factor IX were prescribed annually. This amounts to 1.83 units of FVIII per capita of the population, which is comparable to that of other developed countries. Leading causes of mortality were human immunodeficiency virus-related complications (10 cases) and cerebral bleeding (2 cases). The life expectancy of patients with severe haemophilia in Hong Kong is improving; currently the oldest patient is 60 years old. Such improved survival may be due to enhanced factor availability, prompt treatment of bleeding episodes at home, safer factor products, and better antiviral treatment. Primary prophylaxis is the accepted standard of care for severe and moderate cases, and "Factor First" has become hospital policy. However, 12 patients continue to present treatment challenges, due to the documented presence of factor inhibitors. In all, 28, 100, and 14 cases respectively were positive for human immunodeficiency virus, hepatitis C virus, and hepatitis B virus; the youngest patients with the corresponding infections being 28, 13, and 22 years old. Comprehensive care with dedicated physiotherapy, surgical support, and radionucleotide synovectomy may reduce morbidity further. CONCLUSION: A multidisciplinary approach can further improve the future care for haemophilia patients in Hong Kong.

A bio-activity guided in vitro pharmacokinetic method to improve the quality control of Chinese medicines, application to Si Wu Tang.

The purpose of this study was to demonstrate the feasibility of using a bio-activity guided in vitro pharmacokinetic (BAPK) method in identifying relevant (absorbable and bioactive) markers for quality control (QC) of Chinese medicines (CM), using Si Wu Tang (SWT), a popular CM for women's health, as an example. A stepwise BAPK approach was utilized for relevant marker determination and evaluating of six SWT products: (1) data mining to identify active components of SWT, (2) quantification of the identified active components in each SWT product, (3) determination of in vitro dissolution and metabolism of the components under simulated gastrointestinal conditions, (4) identification of absorbable components or marker(s) via in vitro Caco-2 cell model, (5) stability testing of the permeable marker(s). Our results showed considerable variations in the amount of active components in different SWT products. Of the nine active components identified from data mining, three (ferulic acid, ligustilide, senkyunolide A) were found to be well permeated and stable over three months. Paeoniflorin, the marker designated by Chinese Pharmacopoeia, was poorly permeable and thus could not be considered a relevant marker for SWT. Our preliminary evaluation of the BAPK method appears to be feasible and may offer as a useful approach for identifying relevant markers of other TCM products in the future.

Initial presentation and management of osteosarcoma, and its impact on disease outcome.

OBJECTIVE: To evaluate the initial presenting symptoms and management of osteosarcoma in Hong Kong Chinese children, in relation to any possible impact on disease outcomes. DESIGN: Retrospective study. SETTING: A tertiary referral centre of bone cancer in a university teaching hospital in Hong Kong. PATIENTS: All children aged younger than 18 years with a diagnosis of osteosarcoma who received treatment from March 1994 to October 2005. RESULTS: A total of 51 children were studied. The median age of onset was 13 (range, 3-20) years; 61% were males. The tumours were located in the distal femur and proximal tibia, which accounted for 45% and 22% of the cases, respectively; 24% of patients had metastatic disease at presentation. Swelling (76%) and pain (90%) were the most common presenting complaints. Approximately one third of the patients had a preceding history of trauma. The median duration of initial symptoms to first medical consultation of any sort was 30 (range, 0-360) days. The median time from the first consultation to a definitive diagnosis was 21 (range, 0-350) days; 25% were diagnosed more than 52 days after presentation. Bonesetters were initially consulted by 37% of these patients. From presentation to diagnosis, the median duration was 61 (range, 4-361) days. Analysis of the duration of pre-diagnosis symptoms did not correlate significantly with the development of metastatic disease, response to chemotherapy, feasibility of limb salvage surgery, relapse rates, or survival rates. CONCLUSIONS: In Hong Kong, initial consultation to bonesetters was common. A relatively long delay in between symptom onset and diagnosis of osteosarcoma was encountered. The public and medical practitioners should be made aware of this disease, especially in adolescents.

Magnetic resonance imaging assessment of cardiac and liver iron load in transfusion dependent patients.

BACKGROUND: Magnetic resonance imaging (MRI) is an emerging tool to assess organ-specific iron load in patients with transfusion dependent anemia. OBJECTIVE: We performed MRI T2 star (T2*) assessment in 44 transfusion dependent patients to study the prevalence of cardiac and liver iron overload and the relationship of T2* measurement with various clinical and biochemical parameters. RESULT: Mean age of the study subjects was 19.9 years (range 8.8-32.3) and the mean cardiac T2* was 23.4 +/- 13.8 msec. Fifty percent of the subjects had abnormal cardiac T2* (below 20 msec). Cardiac T2* was not found to have any correlation with serum ferritin or liver T2*. Liver T2* value was abnormal in 79% of the subjects and it correlated inversely with both current and 12 months average serum ferritin (r = -0.44, P = 0.003; r = -0.46, P = 0.002). Clinical parameters including age, duration of transfusion, age starting iron chelation therapy, and ratio between transfusion volume and desferrioxamine dosage were not correlated with cardiac and liver T2*. CONCLUSION: We conclude that iron overload in heart and liver is common in our transfusion dependent patients. Liver T2* has inverse correlation with serum ferritin. Cardiac T2* does not have any correlation with the various clinical and biochemical parameters.

Haematopoietic stem cell transplantation for thalassaemia in Chinese patients.

Beta thalassaemia major is a common hereditary haematological disease in southern Chinese. Advances in transfusion and iron chelation improve survival but haematopoietic stem cell transplantation (HSCT) is still the only curative treatment. Due to repeated blood transfusion and iron overload, thalassaemia patients undergoing HSCT are at a higher risk of graft rejection and transplant-related mortality. The prognostic factors identified to be affecting transplant outcome include hepatomegaly, hepatic fibrosis, and compliance to chelation therapy. Patients can be classified into three classes and conditioning regimens are modified according to the risk. Early stage patients have 85 to 90% chance of disease-free survival, whereas advance stage only has 60% disease-free survival. Mixed chimerism is common after HSCT but majority have satisfactory erythropoiesis without need for further transfusion. Sibling cord blood and bone marrow transplantation has similar outcome. Recently alternative donor transplant has been performed in patients without human leukocyte antigen (HLA)-identical siblings. The result of unrelated-donor bone marrow transplantation is in general inferior but extended HLA matching may improve outcome. The use of unrelated cord blood transplant from a single-centre study showed promising result. The survivors require iron depletion to remove excessive iron store and some may require hormonal replacement therapy. Most of the patients have good quality of life after successful HSCT.

Simultaneous quantification of active components in the herbs and products of Si-Wu-Tang by high performance liquid chromatography-mass spectrometry.

Si-Wu-Tang (SWT), comprising Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular Traditional Chinese Medicine (TCM) formulae for woman's health. Data mining from the available Chinese and English literatures indicated that the major bioactive components of SWT consist of paeoniflorin, paeonol, gallic acid, ferulic acid, Z-ligustilide, ligustrazine, butylphthalide, senkyunolide A and catalpol. Since content determination of the marker compounds is generally considered as an initial step for quality control of TCM product, a high performance liquid chromatography-mass spectrometric method employing both positive and negative electrospray ionization was developed for the simultaneous determination of the nine identified compounds in the raw herbs and products of SWT. The LOQ of the developed assay method for the tested components was 10ng/ml for ligustrazine, 200ng/ml for catalpol, and 100ng/ml for the other seven compounds. The intra-day and inter-day variations of the current assay were within 17.5%. Paeoniflorin, ferulic acid, gallic acid, Z-ligustilide and senkyunolide A were found in all SWT products investigated. Variations in the contents of the studied compounds were observed among batches of raw herbs and SWT products. The currently developed method provides a sensitive and rapid quantification approach that can be useful in the quality control of raw herbs and products of SWT.

Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

Functional characterization and cloning of amino acid transporter B(0,+) (ATB(0,+)) in primary cultured rat pneumocytes.

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I-like cells are described. Asymmetry and activator ion dependency of (3)H-L-arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of (3)H-L-arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated (3)H-L-arginine flux in the apical-to-basolateral and basolateral-to-apical directions. Full length cDNA of rat amino acid transporter B(0,+) (rATB(0,+)) was cloned and its relative expression level studied. Results indicate that uptake of (3)H-L-arginine from apical fluid is dependent on Na(+) and Cl(-). Zwitterionic and cationic amino acids (excluding L-proline and anionic amino acids) inhibited uptake of (3)H-L-arginine from apical, but not basolateral incubation fluid. Apical-to-basolateral transepithelial flux of (3)H-L-arginine was 20x higher than basolateral-to-apical transport. Kinetic studies of (3)H-L-arginine uptake from apical fluid revealed maximal velocity (V(max)) and Michaelis-Menten constants (K(t)) of 33.32 +/- 2.12 pmol/mg protein/15 min and 0.50 +/- 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 +/- 0.16 with Na(+) and 1.11 +/- 0.13 with Cl(-). Expression of rATB(0,+) mRNA was identified by RT-PCR and Northern analysis. Corresponding cloned 3.2 kb rATB(0,+) cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB(0,+) homologues. We conclude that rat pneumocytes express ATB(0,+), which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid.

Effect of application duration of 2% lidocaine jelly on aqueous lidocaine concentration for topical anesthesia in cataract surgery.

BACKGROUND: To evaluate the intraocular penetration of lidocaine 2% gel after different duration of application and to assess their effects on intraoperative pain. METHODS: Samples of aqueous humor were obtained in 41 eyes that had phacoemulsification under topical anesthesia with 2% lidocaine gel. The duration between lidocaine gel application and paracentesis was recorded. The aqueous concentration of lidocaine was measured using high performance liquid chromatography (HPLC). Patients were also asked to report the severity of intraoperative pain using a verbal analogue scale. Correlation analysis was performed to analyze the relationship between duration of lidocaine gel application, aqueous lidocaine concentration and intraoperative pain score. RESULTS: The mean duration of lidocaine gel application was 20.3+/-6.1 min and the mean aqueous lidocaine level was 14.2+/-8.5 micro g/ml. Longer duration of lidocaine gel application was significantly associated with higher aqueous concentration of lidocaine (Spearman's rho=0.58, P<0.001). Eyes in which lidocaine gel was applied for 20 minutes or more had significantly higher aqueous lidocaine level (two-tailed t-test, P=0.001) However, the duration of lidocaine gel application and the aqueous concentration of lidocaine showed no correlation with the patent's perceived pain score (P=0.20 and P=0.79, respectively). CONCLUSIONS: Increase in duration of lidocaine gel application resulted in higher aqueous concentration of lidocaine. Patients' level of perceived pain during phacoemulsification under lidocaine gel anesthesia was not related to the intraocular lidocaine absorption level after 10 min of gel application.