Fix Your Membrane Receptor Imaging: Actin Cytoskeleton and CD4 Membrane Organization Disruption by Chemical Fixation

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artifact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artifacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy

Health services research in the public healthcare system in Hong Kong: An analysis of over 1 million antihypertensive prescriptions between 2004-2007 as an example of the potential and pitfalls of using routinely collected electronic patient data

<b>Objectives</b> Increasing use is being made of routinely collected electronic patient data in health services research. The aim of the present study was to evaluate the potential usefulness of a comprehensive database used routinely in the public healthcare system in Hong Kong, using antihypertensive drug prescriptions in primary care as an example.<p></p> <b>Methods</b> Data on antihypertensive drug prescriptions were retrieved from the electronic Clinical Management System (e-CMS) of all primary care clinics run by the Health Authority (HA) in the New Territory East (NTE) cluster of Hong Kong between January 2004 and June 2007. Information was also retrieved on patients’ demographic and socioeconomic characteristics, visit type (new or follow-up), and relevant diseases (International Classification of Primary Care, ICPC codes). <p></p> <b>Results</b> 1,096,282 visit episodes were accessed, representing 93,450 patients. Patients’ demographic and socio-economic details were recorded in all cases. Prescription details for anti-hypertensive drugs were missing in only 18 patients (0.02%). However, ICPC-code was missing for 36,409 patients (39%). Significant independent predictors of whether disease codes were applied included patient age > 70 years (OR 2.18), female gender (OR 1.20), district of residence (range of ORs in more rural districts; 0.32-0.41), type of clinic (OR in Family Medicine Specialist Clinics; 1.45) and type of visit (OR follow-up visit; 2.39). <p></p> In the 57,041 patients with an ICPC-code, uncomplicated hypertension (ICPC K86) was recorded in 45,859 patients (82.1%). The characteristics of these patients were very similar to those of the non-coded group, suggesting that most non-coded patients on antihypertensive drugs are likely to have uncomplicated hypertension. <p></p> <b>Conclusion</b> The e-CMS database of the HA in Hong Kong varies in quality in terms of recorded information. Potential future health services research using demographic and prescription information is highly feasible but for disease-specific research dependant on ICPC codes some caution is warranted. In the case of uncomplicated hypertension, future research on pharmaco-epidemiology (such as prescription patterns) and clinical issues (such as side-effects of medications on metabolic parameters) seems feasible given the large size of the data set and the comparability of coded and non-coded patients

Examination of the Cytotoxic and Embryotoxic Potential and Underlying Mechanisms of Next-Generation Synthetic Trioxolane and Tetraoxane Antimalarials

Semisynthetic artemisinin-based therapies are the first-line treatment for P. falciparum malaria, but next-generation synthetic drug candidates are urgently required to improve availability and respond to the emergence of artemisinin-resistant parasites. Artemisinins are embryotoxic in animal models and induce apoptosis in sensitive mammalian cells. Understanding the cytotoxic propensities of antimalarial drug candidates is crucial to their successful development and utilization. Here, we demonstrate that, similarly to the model artemisinin artesunate (ARS), a synthetic tetraoxane drug candidate (RKA182) and a trioxolane equivalent (FBEG100) induce embryotoxicity and depletion of primitive erythroblasts in a rodent model. We also show that RKA182, FBEG100 and ARS are cytotoxic toward a panel of established and primary human cell lines, with caspase-dependent apoptosis and caspase-independent necrosis underlying the induction of cell death. Although the toxic effects of RKA182 and FBEG100 proceed more rapidly and are relatively less cell-selective than that of ARS, all three compounds are shown to be dependent upon heme, iron and oxidative stress for their ability to induce cell death. However, in contrast to previously studied artemisinins, the toxicity of RKA182 and FBEG100 is shown to be independent of general chemical decomposition. Although tetraoxanes and trioxolanes have shown promise as next-generation antimalarials, the data described here indicate that adverse effects associated with artemisinins, including embryotoxicity, cannot be ruled out with these novel compounds, and a full understanding of their toxicological actions will be central to the continuing design and development of safe and effective drug candidates which could prove important in the fight against malaria

Entry of Human Papillomavirus Type 16 by Actin-Dependent, Clathrin- and Lipid Raft-Independent Endocytosis

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH

Lump-Sum Bonus Satisfaction: Testing the Construct Validity of a New Pay Satisfaction Dimension

There are both practical and theoretical reasons to measure lump-sum bonus satisfaction. The practical need for such a measure stems from its increased use as a component in modern compensation practices. Based on the means of administering and allocating lump-sum bonuses, a theoretical case can be built suggesting that lump-sum bonus satisfaction constitutes a separate component of pay satisfaction fitting into the Pay Satisfaction Questionaire\u27s (PSQ) theoretical framework. We develop 4 questions that complement the PSQ, and use a series of techniques to test the convergent and discriminant validity of the measure. Empirical evidence shows that bonus-related items are more related to the lump-sum bonus satisfaction measure than other PSQ dimensions. We also demonstrate that the dimension of lump-sum bonus satisfaction has a substantive relationship with attitudinal variables beyond that provided by pay level variables and the PSQ. The development of this measure should foster greater accuracy when assessing pay satisfaction levels and the effects of lump-sum bonus pay policies

Placental transfusion: a review

Recently there have been a number of studies and presentations on the importance of providing a placental transfusion to the newborn. Early cord clamping is an avoidable, unphysiologic intervention that prevents the natural process of placental transfusion. However, placental transfusion, although simple in concept, is affected by multiple factors, is not always straightforward to implement, and can be performed using different methods, making this basic procedure important to discuss. Here, we review three placental transfusion techniques: delayed cord clamping, intact umbilical cord milking and cut-umbilical cord milking, and the evidence in term and preterm newborns supporting this practice. We will also review several factors that influence placental transfusion, and discuss perceived risks versus benefits of this procedure. Finally, we will provide key straightforward concepts and implementation strategies to ensure that placental-to-newborn transfusion can become routine practice at any institution

Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing

Hypnosis for treatment of insomnia in school-age children: a retrospective chart review

BACKGROUND: The purposes of this study are to document psychosocial stressors and medical conditions associated with development of insomnia in school-age children and to report use of hypnosis for this condition. METHODS: A retrospective chart review was performed for 84 children and adolescents with insomnia, excluding those with central or obstructive sleep apnea. All patients were offered and accepted instruction in self-hypnosis for treatment of insomnia, and for other symptoms if it was felt that these were amenable to therapy with hypnosis. Seventy-five patients returned for follow-up after the first hypnosis session. Their mean age was 12 years (range, 7–17). When insomnia did not resolve after the first instruction session, patients were offered the opportunity to use hypnosis to gain insight into the cause. RESULTS: Younger children were more likely to report that the insomnia was related to fears. Two or fewer hypnosis sessions were provided to 68% of the patients. Of the 70 patients reporting a delay in sleep onset of more than 30 minutes, 90% reported a reduction in sleep onset time following hypnosis. Of the 21 patients reporting nighttime awakenings more than once a week, 52% reported resolution of the awakenings and 38% reported improvement. Somatic complaints amenable to hypnosis were reported by 41%, including chest pain, dyspnea, functional abdominal pain, habit cough, headaches, and vocal cord dysfunction. Among these patients, 87% reported improvement or resolution of the somatic complaints following hypnosis. CONCLUSION: Use of hypnosis appears to facilitate efficient therapy for insomnia in school-age children

Postgraduate education for Chinese medicine practitioners: a Hong Kong perspective

<b>Background</b> Despite Hong Kong government's official commitment to the development of traditional Chinese medicine (TCM) over the last ten years, there appears to have been limited progress in public sector initiated career development and postgraduate training (PGT) for public university trained TCM practitioners. Instead, the private TCM sector is expected to play a major role in nurturing the next generation of TCM practitioners. In the present study we evaluated TCM graduates' perspectives on their career prospects and their views regarding PGT.<p></p> <b>Method</b> Three focus group discussions with 19 local TCM graduates who had worked full time in a clinical setting for fewer than 5 years. <p></p> <b>Results</b> Graduates were generally uncertain about how to develop their career pathways in Hong Kong with few postgraduate development opportunities; because of this some were planning to leave the profession altogether. Despite their expressed needs, they were dissatisfied with the current quality of local PGT and suggested various ways for improvement including supervised practice-based learning, competency-based training, and accreditation of training with trainee involvement in design and evaluation. In addition they identified educational needs beyond TCM, in particular a better understanding of western medicine and team working so that primary care provision might be more integrated in the future. <p></p> <b>Conclusion</b> TCM graduates in Hong Kong feel let down by the lack of public PGT opportunities which is hindering career development. To develop a new generation of TCM practitioners with the capacity to provide quality and comprehensive care, a stronger role for the government, including sufficient public funding, in promoting TCM graduates' careers and training development is suggested. Recent British and Australian experiences in prevocational western medicine training reform may serve as a source of references when relevant program for TCM graduates is planned in the futur