Mitochondrial toxicity of aluminium nanoparticles in comparison to its ionic form on isolated rat brain mitochondria.

OBJECTIVES: The aim of this study was to evaluate the toxic effect of AlNPs on rat brain mitochondria and compare it with that of aluminium's ionic form. METHODS: Mitochondria were isolated from rat brain. Isolated mitochondria were treated with normal saline (Control) and different concentrations of aluminium ions (AlIs) and AlNPs (50, 100 and 200 µM). Then, the effect of AlNPs on electron transport chain complexes as well as various endpoints such as mitochondrial oxidative damage (reactive oxygen species, lipid peroxidation, glutathione, and protein carbonyl) and mitochondrial function were assessed. Also, apoptosis was evaluated by cytochrome c release, mitochondrial membrane potential and swelling. RESULTS: When compared to the control group, the exposure to AlNPs showed a marked elevation in oxidative stress markers and inhibition of complex III which was accompanied by disturbance in mitochondrial function. Also, AlNPs induced a significant collapse of mitochondrial membrane potential, mitochondrial swelling, and cytochrome c release. CONCLUSIONS: The comparison of mitochondrial toxicity markers between both forms of aluminium revealed that the toxic effect of AlNPs on isolated brain mitochondria was substantially greater than that that caused by AlIs, which can probably be ascribed to its higher reactivity (Tab. 1, Fig. 8, Ref. 45).

Evaluation of the substitutions in 212, 342 and 215 amino acid positions in binding site of organophosphorus acid anhydrolase using the molecular docking and laboratory analysis.

OBJECTIVE: Organophosphorus Acid Anhydrolase (OPAA) is used as one of the most important enzymes in the decontamination process of organophosphate compounds. In this study, we aimed to evaluate the effects of amino acid substitution in OPAA's substrate-binding site on its catalytic activity using the rational engineering strategy. METHODS: The native and three mutant forms of OPAA enzyme including 4ZWP, 4ZWU and Mut6 were studied using the docking technique toward parathion, paraoxon and R-VX compounds. Furthermore, enzyme assay was performed on the native OPAA and Mut6 toward parathion. RESULTS: Docking results showed a decreased catalytic activity of the mutant forms toward parathion and paraoxon. Furthermore, enzyme assay showed in accordance with docking results a decreased activity of Mut6 compared to the native form. The results of docking prediction for R-VX showed an increased catalytic activity of 4ZWP and 4ZWU. 4ZWU had the highest activity, while the activity of Mut6 was lower than the native form. CONCLUSION: Amino acid positions of 212 and 342 seem to be important sites in the small pocket of OPAA affecting the enzyme catalytic activity. Therefore, substitution of these sites with appropriate amino acids depending on the substrate structure, can affect the enzyme catalytic efficiency (Tab. 2, Fig. 3, Ref. 30).

The effect of synthetic alginate sulfate hydrogels with recombinant PDGF-BB on Wound healing.

OBJECTIVE: Platelet-derived growth factor (PDGF-BB) is an important factor in the regeneration and wound healing. One of the problems is that there is not enough efficiency in the transmission or delivery of such factors in the wound site. In this study, alginate sulfate hydrogel was synthesized with recombinant PDGF-BB growth factor for achieving a quick method in wound healing. METHODS: In this study, Alginate sulfate hydrogel was made by photo-crosslinking method and methacrylate. Its toxicity was evaluated by MTT assay. Transforming growth factor was studied in releasing from synthesized alginate sulfate hydrogels and also, lack of toxicity was confirmed, and hydrogel was made with a recombinant human growth factor. Wounds were created with a diameter of 10 mm on the back of rats in order to check the wound healing. RESULTS: This study showed that alginate sulfate hydrogels-PDGF-BB were faster in wound healing than non-sulfate alginate hydrogels-PDGF-BB. Therefore, the controlled delivery of growth factor system can be a powerful idea for therapeutic applications for wound healing. CONCLUSION: Alginate sulfate hydrogel with recombinant growth factor 4 µg/cm2 (PDGF-BB) was very suitable for wound healing (Tab. 1, Fig. 3, Ref. 34).

Presence of Balamuthia mandrillaris in hot springs from Mazandaran province, northern Iran.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause cutaneous lesions and Balamuthia amoebic encephalitis. The biology and environmental distribution of B. mandrillaris is still poorly understood and isolation of this pathogen from the environment is a rare event. Previous studies have reported that the presence of B. mandrillaris in the environment in Iran may be common. However, no clinical cases have been reported so far in this country. In the present study, a survey was conducted in order to evaluate the presence of B. mandrillaris in hot-spring samples of northern Iran. A total of 66 water samples were analysed using morphological and molecular tools. Positive samples by microscopy were confirmed by performing PCR amplification of the 16S rRNA gene of B. mandrillaris. Sequencing of the positive amplicons was also performed to confirm morphological data. Two of the 66 collected water samples were positive for B. mandrillaris after morphological and molecular identification. Interestingly, both positive hot springs had low pH values and temperatures ranging from 32 °C to 42 °C. Many locals and tourists use both hot springs due to their medicinal properties and thus contact with water bodies containing the organism increases the likelihood of infection. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from hot-spring sources related to human activity. Therefore, B. mandrillaris should be considered as a possible causative agent if cases of encephalitis are suspected following immersion in hot springs in addition to Acanthamoeba and Naegleria.

Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink.

The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 µg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.

[Malignant transformation of plantar ulcers in leprosy: experience of National Leprosy Center in Casablanca]. / Dégénérescence carcinomateuse des maux perforants plantaires d'origine lépreuse: expérience du Centre national de léprologie de Casablanca.

Malignant degeneration is a late complication of plantar ulcers in leprosy. The objective of this study is to describe and analyze the epidemiological, clinical, and therapeutic aspects of this complication in Morocco. A retrospective study was conducted from January 2000 to December 2009 at the National Center of Leprosy (CNL) in Casablanca. All our patients had a histological confirmation. Ten patients were included in this study. There were seven men and three women, with a mean age of 58.8 years. Six patients had a multibacillary form of leprosy and four had a paucibacillary form. The average duration of the plantar ulcers was 34.4 years. Clinical appearance at diagnosis was an ulcerative and vegetative tumor. Treatment was by radical amputation. Evolution was marked by metastatic spread in six patients. One patient died of disseminated disease. In Morocco, leprosy has been on the decline since 1990, but the occurrence of late complications in the leprosy patient as carcinoma of plantar ulcers persists. Prevention of these complications should be part of the national fight against leprosy.

[Familial histoid leprosy]. / Lèpre histoïde familiale.

BACKGROUND: Histoid leprosy is a very particular and rare anatomoclinical form of lepromatous leprosy. Familial cases are extremely rare. We report two new cases of histoid leprosy in a sister and brother born of a non-consanguineous marriage. PATIENTS AND METHODS: We report the case of a sister and brother, aged 21 and 25 years respectively, who were admitted to the National Leprology Centre for multibacillary leprosy. Questioning revealed the existence of family contact with an uncle and two maternal cousins, all of whom had histoid leprosy. On clinical examination, multiple erythematous papules and nodules were noted, some of which were umbilicated while others were ulcerated. Palpation also revealed subcutaneous nodules on the thighs and forearms. No neurological disorders were found in the two patients. Histopathological analysis of cutaneous biopsy samples of nodular lesions for the two patients showed dermal-hypodermic nodular infiltrates comprising spindle cells with a few macrophages. Ziehl-Neelsen staining showed numerous alcohol-acid resistant bacilli (BAAR) without globi. Both patients were placed on the multi-drug regimen recommended by the WHO for multibacillary leprosy. An extremely favourable therapeutic response was obtained after 3 months of treatment. The patients had presented no relapse of erythema nodosum leprosum after two years of follow-up. CONCLUSION: Histoid leprosy is a clinical entity that must be clearly differentiated through its clinical and histopathological features, which distinguish it from polar lepromatous leprosy. There may be a genetic predisposition to this clinical leprosy form, but this requires confirmation by genetic studies.

[Histoid leprosy in Morocco: retrospective study of 18 cases]. / Lèpre histoïde au Maroc: etude rétrospective de 18 cas.

INTRODUCTION: Histoid leprosy is a special and rare anatomoclinical form of multibacillary leprosy. Most of the few large series describing this entity In literature have been reported from India. The purpose of this study was to analyze the epidemiological, clinical and therapeutic characteristics of patients with histoid leprosy in Morocco. MATERIAL AND METHODS: This retrospective study was conducted at the National Centre of Leprology (CNL) in Casablanca from January 1991 to December 2006. Only histologially confirmed cases of histoid leprosy were included. Epidemiological, clinical, histological and therapeutic characteristics were compiled from records and analyzed using EPI-Info version 6. RESULTS: Confirmed histoid leprosy accounted for 18 of the new cases of leprosy recorded at CNL recorded during the 16-year study period. There were 13 men and 5 women with a mean age of 35.2 years. Family exposure was noted in 9 patients (50%) including two who had more than two relatives with leprosy. Nodules/subcutaneous nodules were the most common morphological pattern (88.8%). Neurological involvement was found in 10 patients. Seven patients had grade 1 deformities. Slit skin smears from histoid lesions revealed abundant bacilli with a high bacteriological index ranging from 3 + to 5 + according to the Ridley index. All patients received antibiotic treatment according to the Moroccan protocol. Outcome was favorable in all patients. Six patients (33.3%) developed erythema nodosum leprosum during the course of disease. With a average follow-up of 9 years, no recurrence has been observed. DISCUSSION: Because of its multibacillary character and despite its rarity, histoid leprosy poses a challenge to the leprosy eradication in Morocco. Like other forms of multibacillary leprosy, histoid leprosy requires early detection and prompt multidrug therapy. These requirments should be made a priority in the national program against leprosy.

Effect of student's empowerment program on brucellosis prevention: an application of extended health belief model.

BACKGROUND: To determine the effect of student's empowerment program using the extended health belief model on the brucellosis prevention in rural high school in Divandarreh, Kurdistan province, Iran. MATERIALS AND METHODS: Quasi-experimental study with repeated measure (pre-test, post-test and at 2-month follow-up). In all 220 rural high school students were selected using a cluster sampling method. The data collection tool was a questionnaire based on the Health Belief Model (HBM). Five 1.5 hours sessions using lecture, group discussion, inquiry method, leaflet delivery, and the use of slides with Overhead projector and PowerPoints slide show, designed according to the Systematic Comprehensive Health Education and Promotion Model (SHEP), was presented for intervention group twice a week in schools. Data were analyzed by SPSS 18, using descriptive statistics as well as Chi-square, independent t-test and repeated measures at a significant level less than 0.05. RESULTS: The total mean age of participants was 14.6 ± 2.3. The intervention and control groups had no significant differences in terms of age, gender, and other demographic variables. There was no significant differences in the intervention and control groups before the intervention in terms of awareness, severity, benefits, barriers and self-efficacy. After educational program scores of awareness, severity, susceptibility, benefits, barriers and self-efficacy, and performance were higher in the intervention group compared to the control group. CONCLUSION: Overall, implementation of the educational intervention based on theories and models had good effects on people that are in the risk of infection and zoonotic disease.

Social capital and related factors in Western Iran students.

BACKGROUND AND AIM: Social capital involves a set of norms available in social systems which improves the interaction between people and reduces the costs of interactions and communications. The purpose of this research was to study the social capital of the students of Kurdistan University of Medical Sciences and its related factors, 2017. MATERIAL AND METHODS: The method of this study was the descriptive survey. The statistical sample of this study included all students of Kurdistan University of Medical Sciences among whom 378 students were selected randomly as the study sample. Delaviz questionnaire of social capital was employed for data collection and data analysis was performed using SPSS software, through descriptive statistics (mean, standard deviation) and Inferential statistics (U-Mann Whitney, Kruskal-Wallis). RESULTS: The mean of social capital was 70.56 ± 10/88. The minimum and maximum mean was measured for "participation in local community" and "group participation", respectively. In this study, there were significant associations between social capital and gender, field of study as well as marital status (p < 0/01). CONCLUSIONS: Social capital is known as the most important factor for people dealing with stressful situations, and can facilitate toleration of problems for them. It can also support the health and life satisfaction of students.

Tympanoplasty type-1. Endaural or postaural approach. Should the patient decide?

OBJECTIVE: To examine the preferred patient approach to chronic suppurative otitis media (CSOM) of the safe type. METHODS: We designed a prospective study to note the variables in the patient's initial choice for approach to CSOM of the safe type at Buraidah Central Hospital, Al-Qassim, Saudi Arabia from January 2003 to December 2004. RESULTS: During the 24-month study period, we included 63 consecutive patients diagnosed with CSOM of the safe type. We excluded patients with perforations associated with cholesteatomata, polyps, and acute infections. We administered a questionnaire discussing the options for the operation, and found most males preferred the endaural approach, and most females preferred the postaural approach. CONCLUSION: There was a significant preference by younger age groups for the postaural approach, especially younger females. They were more concerned about the site, visibility, and cosmetic result from the scar, and could camouflage the scar behind the pinna by hair or a traditional headscarf.

Increasing incidence of adenocarcinoma lung in India: Following the global trend?

BACKGROUND: Lung cancer is one of the most common malignant neoplasms worldwide and accounts for more deaths than any other cancer. The clinicopathological profile of lung cancer has shown marked regional and geographical variation. AIMS: We aimed to compare the demographic and pathological profile of lung cancer patients from North India with other Indian and International series. SETTING AND DESIGN: A retrospective study over a period of 5 years from January 2008 to May 2013 was conducted in the Department of Pulmonary Medicine and Sleep Disorders, All India Institute of Medical Sciences, New Delhi. PATIENTS AND METHODS: A total of 397 newly diagnosed patients with lung cancer from January 2008 to May 2013 were included in the study. The clinical, demographic, and pathological features were reviewed and compared with other major National and International reports. Data were entered and analyzed using SPSS software (SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc. RESULTS: A total of 397 patients (86% men, mean age 57.8 years) were studied. The ratio of men to women was 7.4. Majority of patients (78.3%) were current/previous smokers. Small cell carcinoma was diagnosed in 14.6% (58) of patients while 85.4% (339) had nonsmall cell lung carcinoma (NSCLC). Within NSCLC, the most common histology types were squamous cell carcinoma (30%), followed closely by adenocarcinoma (ADC) (28.3%) and large cell carcinoma (1.7%). Majority (87%) of the patient were staged III and IV. About 30.1% patients received anti-tubercular treatment during the current episode before a diagnosis of lung cancer was made. CONCLUSION: The clinicopathological profile of lung cancer has undergone noticeable changes over the last four decades, especially in the increase in ADC incidence and their frequent presence in smokers. Lung cancer is often mistreated as tuberculosis in the Indian subcontinent and hence continues to be diagnosed late.

Ageing, deep vein thrombosis and male gender predict poor outcome after acute Achilles tendon rupture.

AIMS: Patients with an acute Achilles tendon rupture (ATR) take a long time to heal, have a high incidence of deep vein thrombosis (DVT) and widely variable functional outcomes. This variation in outcome may be explained by a lack of knowledge of adverse factors, and a subsequent shortage of appropriate interventions. PATIENTS AND METHODS: A total of 111 patients (95 men, 16 women; mean age 40.3, standard deviation 8.4) with an acute total ATR were prospectively assessed. At one year post-operatively a uniform outcome score, Achilles Combined Outcome Score (ACOS), was obtained by combining three validated, independent, outcome measures: Achilles tendon Total Rupture Score, heel-rise height test, and limb symmetry heel-rise height. Predictors of ACOS included treatment; gender; age; smoking; body mass index; time to surgery; physical activity level pre- and post-injury; symptoms; quality of life and incidence of DVT. RESULTS: There were three independent variables that correlated significantly with the dichotomised outcome score (ACOS), while there was no correlation with other factors. An age of less than 40 years old was the strongest independent predictor of a good outcome one year after ATR (odds ratio (OR) 0.20, 95% confidence interval (CI) 0.08 to 0.51), followed by female gender (OR) 4.18, 95% CI 1.01 to 17.24). Notably, patients who did not have a DVT while immobilised post-operatively had a better outcome (OR 0.31, 95% CI 0.12 to 0.80). CONCLUSION: Over the age of 40 years, male gender and having a DVT while immobilised are independent negative predictors of outcome in patients with an acute ATR. Cite this article: Bone Joint J 2016;98-B:1635-41.

Intermittent pneumatic compression reduces the risk of deep vein thrombosis during post-operative lower limb immobilisation: a prospective randomised trial of acute ruptures of the Achilles tendon.

Deep vein thrombosis is a common complication of immobilising the lower limb after surgery. We hypothesised that intermittent pneumatic compression (IPC) therapy in outpatients who had undergone surgical repair of acute ruptures of the Achilles tendon could reduce the incidence of this problem. A total of 150 patients who had undergone surgical repair of the Achilles tendon were randomised to either treatment with IPC for six hours per day (n = 74) under an orthosis or treatment as usual (n = 74) in a plaster cast without IPC. At two weeks post-operatively, the incidence of deep vein thrombosis was assessed using blinded, double-reported compression duplex ultrasound. At this point, IPC was discontinued and all patients were immobilised in an orthosis for a further four weeks. At six weeks post-operatively, a second compression duplex ultrasound scan was performed. At two weeks, the incidence of deep vein thrombosis was 21% in the treated group and 37% in the control group (p = 0.042). Age over 39 years was found to be a strong risk factor for deep vein thrombosis (odds ratio (OR) = 4.84, 95% confidence interval (CI) 2.14 to 10.96). Treatment with IPC, corrected for age differences between groups, reduced the risk of deep vein thrombosis at the two-week point (OR = 2.60; 95% CI 1.15 to 5.91; p =0.022). At six weeks, the incidence of deep vein thrombosis was 52% in the treated group and 48% in the control group (OR 0.94, 95% CI 0.49 to 1.83). IPC appears to be an effective method of reducing the risk of deep vein thrombosis in the early stages of post-operative immobilisation of outpatients. Further research is necessary to elucidate whether it can confer similar benefits over longer periods of immobilisation and in a more heterogeneous group of patients.

Can foot compression under a plaster cast prevent deep-vein thrombosis during lower limb immobilisation?

We hypothesised that adjuvant intermittent pneumatic compression (IPC) beneath a plaster cast would reduce the risk of deep-vein thrombosis (DVT) during post-operative immobilisation of the lower limb. Of 87 patients with acute tendo Achillis (TA) rupture, 26 were prospectively randomised post-operatively after open TA repair. The treatment group (n = 14) received two weeks of IPC of the foot for at least six hours daily under a plaster cast. The control group (n = 12) had no additional treatment. At two weeks post-operatively all patients received an orthosis until follow-up at six weeks. At two and six weeks the incidence of DVT was assessed using colour duplex sonography by two ultrasonographers blinded to the treatment. Two patients withdrew from the study due to inability to tolerate IPC treatment. An interim analysis demonstrated a high incidence of DVT in both the IPC group (9 of 12, 75%) and the controls (6 of 12, 50%) (p = 0.18). No significant differences in incidence were detected at two (p = 0.33) or six weeks (p = 0.08) post-operatively. Malfunction of the IPC leading to a second plaster cast was found to correlate with an increased DVT risk at two weeks (ϕ = 0.71; p = 0.019), leading to a premature abandonment of the study. We cannot recommend adjuvant treatment with foot IPC under a plaster cast for outpatient DVT prevention during post-operative immobilisation, owing to a high incidence of DVT related to malfunctioning of this type of IPC application.

Immune thrombocytopenic purpura in adults in the last 10 years: single-centre experience.

BACKGROUND: Immune thrombocytopenic purpura (ITP) is a benign disease with low morbidity and mortality and frequent remissions that occur spontaneously or in response to first-line treatment with steroids or splenectomy. AIM: The purpose of this study is to describe the clinical outcomes of 170 patients with ITP diagnosed and/or treated in our hospital between 2000 and 2010. METHODS AND RESULTS: The median age at diagnosis was 47 years. Forty three (25%) were asymptomatic, 65% had minor skin or mucosal bleeding and 10% had significant bleeding from the gastrointestinal or genitourinary system. The median platelet count at diagnosis was 13x10(9)/L (range: 0-98x10(9)/L). Median follow-up of all patients was 13 months. Ninety-five patients had a follow-up longer than 12 months, with median 44 months (range 14-384). Corticosteroids were the initial treatment for 161/170 (95%) patients, 38 (22%) were splenectomized, 25 (14.7%) were treated with intravenous gamma globulins, while 9 did not received any specific treatment. A complete response to initial treatment (prednisone±splenectomy) was achieved in 55/161 (34.2%), a partial response in 90 (55.9%) and no response in 16 (9.9%) patients. In the group of patients with follow-up longer than 1 year; 28 (29%) patients had refractory or unresponsive ITP with a median follow-up of 66 months. All patients with refractory ITP were treated with steroids, 11 were splenctomized, significantly more patients with refractory ITP 12 (43%) were treated with IVIG compared with other ITP patients (16%), p=0.005. The median age of 38 splenectomized patients was 28 years and it is significantly different from the other patients (p<0.001). There were no significant differences in other characteristics between splenctomized or refractory ITP and other patients at diagnosis. CONCLUSION: Our results were similar to results already reported in other similar studies.

Bilateral congenital choanal atresia presenting at age twenty two years: an unusual case report.

Choanal atresia is a congenital absence of communication between the nasal cavity and nasopharynx. Bilateral Choanal Atresia usually present immediately after birth and in the neonatal period. We report an unusual case who presented at the age of twenty two years. This case was successfully managed by a transnasal approach. Choanal atresia should be considered as a rare diagnostic possibility in any patient who presents with total nasal obstruction and persistent mouth breathing.

Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa.

The virulence of the opportunistic pathogen Pseudomonas aeruginosa is largely dependent upon the extracellular production of a number of secreted proteins with toxic or degradative activities. The synthesis of several exoenzymes is controlled in a cell-density-dependent manner by two interlinked quorum-sensing systems. Their secretion across the outer membrane occurs through the Xcp translocation machinery. The xcp locus located at 40 min on the chromosome consists of two divergently transcribed operons, namely xcpPQ and xcpR to xcpZ. In this study, transcriptional fusions were constructed between the xcpP and xcpR genes and the lacZ reporter. Transcriptional activation of the xcpP and xcpR genes in P. aeruginosa is growth-phase dependent and the lasR-lasI autoinduction system is required for this control. In the heterologous host Escherichia coli, the lasR gene product, together with its cognate autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), activates both the xcpP-lacZ and the xcpR-lacZ gene fusion. The second P. aeruginosa quorum-sensing modulon rhIR-rhII (vsmR-vsmI) is also involved in the control of the xcp genes. Expression of the lacZ fusions is strongly reduced in PANO67, a pleiotropic mutant defective in the production of N-acyl-homoserine lactones responsible for the activation of RhIR. Furthermore, introduction of the lasR mutation in PANO67 results in additional diminution of xcpR transcription, indicating that the two systems can regulate their target genes independently. These data demonstrate that expression of the xcp secretion system depends on a complex regulatory network involving cell-cell signalling which controls production and secretion of virulence-associated factors.

The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase.

The global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-L-homoserine lactone (BHL), of the cognate transcriptional activator RhIR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhIR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhIR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhIR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-L-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhIR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.

A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS.

In Pseudomonas aeruginosa, the production of many virulence factors and secondary metabolites is regulated in concert with cell density through quorum sensing. Two quorum-sensing regulons have been identified in which the LuxR homologues LasR and RhlR are activated by N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) respectively. The lasR and rhlR genes are linked to the luxl homologues lasl and rhll, which are responsible for synthesis of OdDHL and BHL, respectively. As lasRl and rhlRl are both involved in regulating synthesis of exoenzymes such as elastase, we sought to determine the nature of their interrelationship. By using lacZ transcriptional fusions in both homologous (P. aeruginosa) and heterologous (Escherichia coli) genetic backgrounds we provide evidence that (i) lasR is expressed constitutively throughout the growth cycle, (ii) rhlR expression is regulated by LasR/OdDHL, and (iii) that RhlR/BHL regulates rhll. We also show that expression of the stationary-phase sigma factor gene rpoS is abolished in a P. aeruginosa lasR mutant and in the pleiotropic BHL-negative mutant PANO67. Furthermore, our data reveal that kin E. coli, an rpoS-lacZ fusion is regulated directly by RhlR/BHL. Taken together, these results indicate that P. aeruginosa employs a multilayered hierarchical quorum-sensing cascade involving RhlR/BHL and LasR/OdDHL, interlinked via RpoS, to integrate the regulation of virulence determinants and secondary metabolites with adaptation and survival in the stationary phase.