N-acylhomoserine lactones antagonize virulence gene expression and quorum sensing in Staphylococcus aureus

Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 muM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.     

Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique

Abstract

Conclusion: In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-β3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue.

Objective: Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-β) and interaction of auricular chondrocytes with HADSCs for new cartilage generation.

Methods: Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-β3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-βs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days.

Results: According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen. The results showed HADSCs test groups (cultured with TGF-β3) displayed chondrocytes-like cells morphology with typical lacunae structure compared to the control group without TGF-β3 after 2 weeks. Additionally, the HADSCs test groups increased in cell viability; an increase in expression of chondrocytes-specific genes (collagen type II, aggrecan core protein, SOX 9 and elastin) compared to the control. This study found that human auricular chondrocytes cells and growth factor had a positive influence in inducing HADSCs chondrogenic effects, in terms of chondrogenic differentiate of feature, increase of cell viability, and up-regulated expression of chondrogenic genes.     

Rheological Performance of Magnetorheological Grease with Embedded Graphite Additives

The use of highly viscous grease as a medium in magnetorheological grease (MRG) provides the benefit of avoiding sedimentation from occurring. However, it limits the expansion of yield stress in the on-state condition, thus reducing the application performance during operation. Therefore, in this study, the improvement in the rheological properties of MRG was investigated through the introduction of graphite as an additive. MRG with 10 wt % graphite (GMRG) was fabricated, and its properties were compared to a reference MRG sample. The microstructure of GMRG was characterized using an environmental scanning electron microscope (ESEM). The rheological properties of both samples, including apparent viscosity, yield stress, and viscoelasticity, were examined using a shear rheometer in rotational and oscillatory modes. The results demonstrated a slight increase in the apparent viscosity in GMRG and a significant improvement in yield stress by 38.8% at 3 A with growth about 32.7% higher compared to MRG from 0 to 3 A. An expansion of the linear viscoelastic region (LVE) from 0.01% to 0.1% was observed for the GMRG, credited to the domination of the elastic properties on the sample. These obtained results were confirmed based on ESEM, which described the contribution of graphite to constructing a more stable chain structure in the GMRG. In conclusion, the findings highlight the influence of the addition of graphite on improving the rheological properties of MRG. Hence, the addition of graphite in MRG shows the potential to be applied in many applications in the near future.     

15d-PGJ2 Induces Apoptosis of MCF-7 and MDA-MB-231 Cells via Increased Intracellular Calcium and Activation of Caspases,Independent of ER and ER

Reports indicate that 15deoxydelta12,14prostaglandinJ2 (15dPGJ2) has anticancer activities, but its mechanisms of action have yet to be fully elucidated We therefore investigated the effects of 15dPGJ2 on the human breast cancer cell lines, MCF7 (estrogen receptor ERER) and MDAMB231 (ERER) Cellular proliferation and cytotoxicity were determined using the 3(4,5dimethylthiazol2yl)2,5diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays while apoptosis was determined by fluorescence microscopy and flow cytometry using annexin Vpropidium iodide (PI) staining ER expression was determined by Western blotting Intracellular calcium was stained with Fluo4 AM while intracellular caspase activities were detected with CaspaseFLICA and measured by flow cytometry We showed that 15dPGJ2 caused a significant increase in apoptosis in MCF7 and MDAMB231 cells ER protein expression was reduced in treated MCF7 cells but preincubation with the ER inhibitor ICI 182 780 did not affect the percentage of apoptotic cells The expression of ER was unchanged in both cell lines In addition, 15dPGJ2 increased intracellular calcium (Ca) staining and caspase 8, 9 and37 activities We therefore conclude that 15dPGJ2 induces caspasedependent apoptosis that is associated with an influx of intracellular Ca with no involvement of ER signaling     

A prospective study of tracheal intubation in an academic emergency department in Malaysia

Background

Airway management is an important part of the management of the critically ill and injured patients in the Emergency Department (ED). Numerous studies from developed countries have demonstrated the competency of emergency doctors in intubation. To date there have been no published data on intubations performed in EDs in Malaysia.

Methods

Data on intubations from 7 August 2007 till 28 August 2008 were prospectively collected.

Results

There were 228 intubations included in the study period. Cardiopulmonary arrest was the main indication for intubation (35.5%). The other indications were head injury (18.4%), respiratory failure (15.4%), polytrauma (9.6%) and cerebrovascular accident (7.0%). All of the 228 patients were successfully intubated. Rapid sequence intubation (RSI) was the most frequent method (49.6%) of intubation. A total of 223 (97.8%) intubations were done by ED personnel. In 79.8% of the cases, intubations were successfully performed on the first attempt. Midazolam was the most common induction agent used (97 patients), while suxamethonium was the muscle relaxant of choice (109 patients). There were 34 patients (14.9%) with 38 reported immediate complications. The most common complication was oesophageal intubation.

Conclusion

Emergency Department UKMMC personnel have a high competency level in intubation with an acceptable complication rate. RSI was the most common method for intubation.     

Evaluation of cardiac and hepatic iron overload in thalassemia major patients with T2* magnetic resonance imaging

Objectives: Recent advancements have promoted the use of T2* magnetic resonance imaging (MRI) in the non-invasive detection of iron overload in various organs for thalassemia major patients. This study aims to determine the iron load in the heart and liver of patients with thalassemia major using T2* MRI and to evaluate its correlation with serum ferritin level and iron chelation therapy.

Methods: This cross-sectional study included 162 subjects diagnosed with thalassemia major, who were classified into acceptable, mild, moderate, or severe cardiac and hepatic iron overload following their T2* MRI results, respectively, and these were correlated to their serum ferritin levels and iron chelation therapy.

Results: The study found that 85.2% of the subjects had normal cardiac iron stores. In contrast, 70.4% of the subjects had severe liver iron overload. A significant but weak correlation (r?=??0.28) was found between cardiac T2* MRI and serum ferritin, and a slightly more significant correlation (r?=?0.37) was found between liver iron concentration (LIC) and serum ferritin.

Discussion: The findings of this study are consistent with several other studies, which show that patients generally manifest with liver iron overload prior to cardiac iron overload. Moreover, iron accumulation demonstrated by T2* MRI results also show a significant correlation to serum ferritin levels.

Conclusion: This is the first study of its kind conducted in Indonesia, which supports the fact that T2* MRI is undoubtedly valuable in the early detection of cardiac and hepatic iron overload in thalassemia major patients.     

Cellular and Molecular Changes in MNU-Induced Breast Tumours Injected with PF4 or bFGF

Background: Angiogenic activity has been considered to reflect important molecular events during breast tumour development. The present study concerned cellular and molecular changes of MNU-induced breast tumours subjected to promotion and suppression of angiogenesis. Methods: Female Sprague Dawley rats at the age of 21 days received MNU at the dose 70 mg/kg of body weight by intraperitoneal injection. Three months post-carcinogen initiation, mammary tumours were palpated and their growth was monitored. When the tumour diameter reached 1.0 ± 0.05 cm, rats were given bFGF or PF4 intratumourally at a dose of 10 μg/tumour. Entire palpable tumour were subsequently excised and subjected to histology examination, IHC staining, and RT-PCR. Results: No critical morphological changes were observed between pro-angiogenic factor, bFGF, and control groups. However, increase of tumour size with more necrotic and diffuse areas was notable in tumours after anti-angiogenic PF4 intervention. ER and PR mRNA expression was significantly up- and down-regulated in bFGF and PF4 groups, respectively. The trends were significantly associated with peri- and intratumoural MVD counts. However, irrespective of whether we promoted or inhibited angiogenesis, the expression of EGFR and ERBB2 continued to be significantly increased but this was not significantly associated with the MVD score. No significant differences in E-cadherin and LR gene expression were noted between intervention and control groups. Conclusion: ER and PR receptor expression shows consistent responses when tumour angiogenesis is manipulated either positively or negatively. Our study adds to current understanding that not only do we need to target hormonal receptors, as presently practiced, but we also need to target endothelial receptors to successfully treat breast cancer.     

Anxiety Disorders in Family Caregivers of Breast Cancer Patients Receiving Oncologic Treatment in Malaysia

Background: Anxiety is recognized as a normal psychological reaction of those caring for cancer patients. However, anxiety disorders in caregivers may interfere with their care-giving role and require further clinical attention. Objectives: To determine the prevalence and associated factors of anxiety disorders among caregivers of breast cancer patients receiving oncologic treatment in Kuala Lumpur Hospital. Methodology: A cross-sectional study was conducted on 130 caregiver-patient dyads, recruited by non-random sampling at Kuala Lumpur Hospital. Data were collected in 2 stages: 1) the caregivers were screened for psychological distress using the Depression, Anxiety, Stress, Scale (DASS-21) while other related factors for the patients and their caregivers were obtained; 2) the identified distressed caregivers (n=64) were then administered the Mini International Neuropsychiatric Interview (MINI) to diagnose anxiety disorders. Results: A total of 11.5% (n=15) of the caregivers reported suffering from anxiety disorders. Bivariate analysis found duration of caregiving (OR=3.31; CI=2.21-11.93), shared caregiving (OR=4.07; CI=1.34-12.36), and patients’ treatment type (OR=3.42; CI=1.92-12.76) were significantly associated with anxiety disorders (p value <0.05), with shared caregiving and patient’s treatment type remaining significant using logistic regression (p value < 0.05, R2 = 0.255). Conclusions: Every one in ten of the caregivers in this study had a diagnosable anxiety disorder, associated with certain care-giving factors and patients’ treatment. This should alert clinicians to such risk and indicates psychological support needs for family caregivers.