Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor

Endocytosis of the epidermal growth factor receptor (EGFR) is important for the regulation of EGFR signaling. However, EGFR endocytosis mechanisms are poorly understood, which precludes development of approaches to specifically inhibit EGFR endocytosis and analyze its impact on signaling. Using a combination of receptor mutagenesis and RNA interference, we demonstrate that clathrin-dependent internalization of activated EGFR is regulated by four mechanisms, which function in a redundant and cooperative fashion. These mechanisms involve ubiquitination of the receptor kinase domain, the clathrin adaptor complex AP-2, the Grb2 adaptor protein, and three C-terminal lysine residues (K1155, K1158, and K1164), which are acetylated, a novel posttranslational modification for the EGFR. Based on these findings, the first internalization-defective EGFR mutant with functional kinase and normal tyrosine phosphorylation was generated. Analysis of the signaling kinetics of this mutant revealed that EGFR internalization is required for the sustained activation of protein kinase B/AKT but not for the activation of mitogen-activated protein kinase.     

The Doctor-Patient Relationship: A Survey of Attitudes and Practices of Doctors in Singapore

This article reports the results of a survey, by mailed questionnaire, of the attitudes, values and practices of doctors in Singapore with respect to the doctor-patient relationship. Questionnaires were sent to a random sample of 475 doctors (261 general practitioners and 214 medical specialists), out of which 249 (52.4%) valid responses were completed and returned. The survey is the first of its kind in Singapore. Questions were framed around issues of medical paternalism, consent and patient autonomy. As the doctors were exposed to Western ethical concepts in their training, we were not surprised to find that they would mostly allow patients some say in decision-making and keep patients reasonably informed. In respecting patient autonomy, they would usually seek to influence patient choice by persuasion. However, the residual 'Asian-ness' of doctors in Singapore gives rise to some inconsistencies between values and practices. Many doctors still believe that a number of their patients are incapable of rational choice. There is some lack of openness in telling patients the whole truth. When patients choose to refuse treatment, many doctors are prepared to involve family members in making a consensus decision.
Doctors were also asked how they made ethical judgements in the face of dilemmas, and how they would like disputes with patients to be resolved. By and large, the doctors prefer to make their own judgements rather than to rely on rules. They also wish to keep the law courts out of disputes with patients, preferring less public ways of settling disputes.     

Failure of Arm Movement Control in Stroke Patients,Characterized by Loss of Complexity

We study the mechanism of human arm-posture control by means of nonlinear dynamics and quantitative time series analysis methods. Utilizing linear and nonlinear measures in combination, we find that pathological tremors emerge in patient dynamics and serve as a main feature discriminating between normal and patient groups. The deterministic structure accompanied with loss of complexity inherent in the tremor dynamics is also revealed. To probe the underlying mechanism of the arm-posture dynamics, we further analyze the coupling patterns between joints and components, and discuss their roles in breaking of the organization structure. As a result, we elucidate the mechanisms in the arm-posture dynamics of normal subjects responding to the gravitational force and for the reduction of the dynamic degrees of freedom in the patient dynamics. This study provides an integrated framework for the origin of the loss of complexity in the dynamics of patients as well as the coupling structure in the arm-posture dynamics.     

Purification and Characterization of Bacteriocin Produced by Weissella confusa A3 of Dairy Origin

A dramatic increase in bacterial resistance towards currently available antibiotics has raised worldwide concerns for public health. Therefore, antimicrobial peptides (AMPs) have emerged as a promisingly new group of therapeutic agents for managing infectious diseases. The present investigation focusses on the isolation and purification of a novel bacteriocin from an indigenous sample of cow milk and it’s mode of action. The bacteriocin was isolated from Weissella confusa A3 that was isolated from the sample and was shown to have inhibitory activity towards pathogenic bacteria namely Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Micrococcus luteus. The bacteriocin was shown to be heat stable and functioned well at low pH (2 to 6). Reduction of activity was shown after treatment with proteinase K, trypsin and peptidase that confirmed the proteinaceous nature of the compound. MALDI-TOF analysis of the sample gave a mass approximating 2.7 kDa. The membrane of the bacteria was disrupted by the bacteriocin causing SYTOX^® green dye to enter the cell and bind to the bacterial DNA giving fluorescence signal. Bacterial cell treated with the bacteriocin also showed significant morphological changes under transmission electron microscope. No virulence and disease related genes can be detected from the genome of the strain.     

Analysis of acoustic stimulus to Liriomyza trifolii (Diptera: Agromyzidae) through Electroantennogram (EAG) technique

The study utilized the Electroantennogram (EAG) technique as biological acoustic stimulus to Liriomyza trifolii (Diptera: Agromyzidae). Pure tone sounds and courtship songs were tested in the excised head of test specimens. Significant EAG stimuli were detected when released at 20 Hz pure tone sound together with the courtship song having long pulse train patterns of Cryptonevra inquilina and Lipara japonica (Chloropidae). For their biological effectiveness, movement behaviors were analyzed with stimulus taken from EAG recordings. No attraction effect was observed even with and without acoustic stimulus. However, movement activities were transformed by information entropy and observed to be clearly different when selected acoustic stimuli were broadcast. The combination of response to EAG and behavioral observation would be useful to determine effective movement modification through acoustic stimulus in insect pests that are necessary toward the development of an acoustic pest management tool in agriculture.     

Validation of a Rapid and Sensitive LC-MS/MS Method for Determination of Exemestane and Its Metabolites, 17β-Hydroxyexemestane and 17β-Hydroxyexemestane-17-O-β-D-Glucuronide: Application to Human Pharmacokinetics Study

A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of exemestane pharmacokinetics and its metabolites, 17β-dihydroexemestane (active metabolite) and 17β-dihydroexemestane-17-O-β-D-glucuronide (inactive metabolite) in human plasma. Their respective D3 isotopes were used as internal standards. Chromatographic separation of analytes was achieved using Thermo Fisher BDS Hypersil C18 analytic HPLC column (100 × 2.1 mm, 5 μm). The mobile phase was delivered at a rate of 0.5 mL/min by gradient elution with 0.1 % aqueous formic acid and acetonitrile. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionisation (ESI) and monitored by multiple reaction monitoring (MRM) in positive mode. Mass transitions 297 > 121 m/z, 300 > 121 m/z, 299 > 135 m/z, 302 > 135 m/z, 475 > 281 m/z, and 478 > 284 m/z were monitored for exemestane, exemestane-d3, 17β-dihydroexemestane, 17β-dihydroexemestane-d3, 17β-dihydroexemestane-17-O-β-D-glucuronide, and 17β-dihydroexemestane-17-O-β-D-glucuronide-d3 respectively. The assay demonstrated linear ranges of 0.4 – 40.0 ng/mL, for exemestane; and 0.2 – 15.0 ng/mL, for 17β-dihydroexemestane and 17β-dihydroexemestane-17-O-β-D-glucuronide, with coefficient of determination (r²) of > 0.998. The precision (coefficient of variation) were ≤10.7%, 7.7% and 9.5% and the accuracies ranged from 88.8 to 103.1% for exemestane, 98.5 to 106.1% for 17β-dihydroexemestane and 92.0 to 103.2% for 17β-dihydroexemestane-17-O-β-D-glucuronide. The method was successfully applied to a pharmacokinetics/dynamics study in breast cancer patients receiving exemestane 25mg daily orally. For a representative patient, 20.7% of exemestane in plasma was converted into 17β-dihydroexemestane and 29.0% of 17β-dihydroexemestane was inactivated as 17β-dihydroexemestane-17-O-β-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells.     

Extent and distribution of tibial osteochondral disruption during simulated landing impact with axial tibial rotation restraint

Post-traumatic knee osteochondral injuries are often coupled with anterior cruciate ligament (ACL) injury mechanisms during landing. However, it is not well understood whether restraining axial tibial rotation during landing would influence the extent and distribution of osteochondral disruption. Using ski landing as an example, this study subjected knee specimens to simulated landing impact without and with axial tibial rotation restraint, and investigated the extent and distribution of osteochondral disruption at the tibial plateau. Twenty-one porcine knee specimens were randomly divided into three test conditions, namely: (1) control, (2) impact only (I), and 3) impact with restraint (IR). Simulated landing impact was applied to the specimens based on a single 10 Hz haversine. Osteochondral explants were obtained from anterior, middle and posterior regions of medial and lateral tibial compartments. The extent of cartilage and trabecular disruption in these explants was examined based on histology, SEM and microCT. Only specimens in unrestrained condition incurred ACL failure upon impact. Restraining axial tibial rotation during simulated impact generally inflicted cartilage damage and deformation, and further caused trabecular disruption. Axial tibial rotation restraint did not necessarily restrict anterior tibial translation, as indicated by the presence of relative posterior femoral translation and osteochondral disruption at anterior–posterior tibial regions. While the results obtained in the current study may not be completely translatable to human models, there is likelihood that restraining axial tibial rotation during landing may help to prevent ACL failure, but will also induce osteochondral disruption in most tibial regions.     

A Comprehensive Review on the Anticancer Potential of Bacteriocin: Preclinical and Clinical Studies

International Journal of Peptide Research and Therapeutics - Chikungunya virus is a major arbovirus of great public health concern in the whole world, but no vaccine is yet available, still advance...