Influence of dissolution on the uptake of bimetallic nanoparticles Au@Ag-NPs in soil organism Eisenia fetida.

A key aspect in the safety testing of metal nanoparticles (NPs) is the measurement of their dissolution and of the true particle uptake in organisms. Here, based on the tendency of Ag-NP to dissolve and Au-NP to be inert in the environment, we exposed the earthworm Eisenia fetida to Au core-Ag shell NPs (Au@Ag-NPs, Ag-NPs with a Au core) and to both single and combined exposures of non-coated Au-NPs, Ag-NPs, Ag+ and Au+ ions in natural soil. Our hypothesis was that the Ag shell would partially or completely dissolve from the Au@Ag-NPs and that the Au core would thereby behave as a tracer of particulate uptake. Au and Ag concentrations were quantified in all the soils, in soil extract and in organisms by inductively coupled plasma mass spectrometry (ICP-MS). The earthworm exposed to Au@Ag-NPs, and to all the combinations of Ag and Au, were analyzed by single particle inductively coupled plasma time-of-flight mass spectrometry (spICP-TOFMS) to allow the quantification of the metals that were truly part of a bimetallic particle. Results showed that only 5% of the total metal amounts in the earthworm were in the bimetallic particulate form and that the Ag shell increased in thickness, suggesting that biotransformation processes took place at the surface of the NPs. Additionally, the co-exposure to both metal ions led to a different uptake pattern compared to the single metal exposures. The study unequivocally confirmed that dissolution is the primary mechanism driving the uptake of (dissolving) metal NPs in earthworms. Therefore, the assessment of the uptake of metal nanoparticles is conservatively covered by the assessment of the uptake of their ionic counterpart.

Introducing holmium laser enucleation of the prostate alongside transurethral resection of the prostate improves outcomes of each procedure.

INTRODUCTION: Holmium laser enucleation of the prostate (HoLEP) is recognised as an alternative to transurethral resection of the prostate (TURP). HoLEP has been demonstrated to be at least as effective as TURP with less morbidity but its introduction to practice has been limited in part by the learning curve of a novel procedure. This study examined the effects of introducing HoLEP alongside an established practice of TURP on early morbidity and length of hospital stay (LOS). METHODS: A retrospective review of all patients who underwent HoLEP and TURP between April 2007 and July 2011 was undertaken. HoLEP was introduced in April 2008; patients undergoing TURP before this were considered as a historical control group. Data were collected concerning resection/enucleation weight, blood transfusions and LOS. RESULTS: Overall, 772 patients underwent HoLEP or TURP within the 52-month study period: 164 underwent TURP prior to the introduction of HoLEP (TURP-A), 425 had TURP after the introduction of HoLEP (TURP-B) and 183 underwent HoLEP. The mean removed weight was 24g (standard deviation [SD]: 21g) for TURP-A, 19g for TURP-B (SD: 16g) and 38g (SD: 32g) for HoLEP (p<0.005). Blood transfusion rates were 5.5%, 2.2% and 1.6% for the TURP-A, TURP-B and HoLEP groups respectively (p<0.05). For TURP-A patients, the mean LOS was 5.6 days (SD: 3.5 days, 95% confidence interval [CI]: 5.3-6.0 days). The mean LOS for TURP-B patients was 4.4 days (SD: 4.4 days, 95% CI: 4.2-4.8 days). HoLEP patients had a mean LOS of 3.0 days (SD: 3.0 days, 95% CI: 2.6-3.4 days). CONCLUSIONS: The introduction of HoLEP alongside TURP is associated with lower rates of blood transfusion and shorter LOS for all patients. This is likely to be due to the use of HoLEP rather than TURP in patients with larger prostates, who are more likely to have complications.

Intracellular sequestration of amiodarone: role of vacuolar ATPase and macroautophagic transition of the resulting vacuolar cytopathology.

BACKGROUND AND PURPOSE: Tissue deposits of the anti-arrhythmic drug amiodarone are a major source of side effects (skin discoloration, etc.). We addressed the mechanism of the concentration of amiodarone in cells, and characterized the resulting vacuolar cytopathology and its evolution towards macroautophagy. EXPERIMENTAL APPROACH: Sequestration of amiodarone in human cells (macrophages, smooth muscle cells, HEK 293a cells) was evaluated using its violet fluorescence and cytopathology using GFP-conjugated subcellular markers. Autophagic signalling was probed by immunoblotting for the effector protein LC3. A patient biopsy of amiodarone-induced blue-gray skin discoloration was investigated for the presence of macroautophagy (immunofluorescence for LC3). KEY RESULTS: Most of the amiodarone (1-20 microM, 4-24 h) captured by cultured cells (macrophages were most avid) was present in enlarged vacuoles. The specific vacuolar ATPase (V-ATPase) inhibitors, bafilomycin A1 or FR167356, prevented vacuolization and drug uptake. Vacuoles in HEK 293a cells were positive for markers of late endosomes and lysosomes (GFP-Rab7, -CD63) and for an effector of macroautophagy, GFP-LC3. The vacuoles accumulated endogenous LC3 and filled with lipids (Nile red staining) following longer amiodarone treatments (> or =24 h). The electrophoretic mobility of both GFP-LC3 and endogenous LC3 changed, showing activation in response to amiodarone. Paraffin tissue sections of the pigmented skin exhibited granular LC3 accumulation in superficial dermis macrophages. CONCLUSION AND IMPLICATIONS: Vacuolar sequestration of amiodarone occurs at concentrations close to therapeutic levels, is mediated by V-ATPase and evolves towards persistent macroautophagy and phospholipidosis. This cytopathology is not cell type specific, but tissue macrophages appear to be particularly susceptible.

Evidence-based guide to perioperative medicine.

The discipline of perioperative medicine is assuming greater importance as increasing numbers of older patients with medical comorbidity undergo complex surgical procedures. If patient outcomes and use of limited hospital resources are to be optimized, physicians with skills and interest in perioperative risk assessment and therapeutic intervention are needed. This systematic review attempts to provide an evidence-based update in several key areas in the management of the perioperative patient.

Acute autoimmune cardiomyopathy in primary antiphospholipid antibody syndrome.

We present a case of acute pulmonary oedema as the first presentation of autoimmune cardiomyopathy in primary antiphospholipid antibody syndrome in a patient who had no previous cardiac history. Five days of methylprednisolone at 500 mg/day followed by 100 mg/day for 10 days and then a weaning course of oral prednisone resulted in effective resolution of the acute diffuse cardiomyopathy. Her cardiac status became clinically and echocardiographically normal. We illustrate the effectiveness of immunosuppressive therapy as an adjunct to standard anti-failure measures in such presentations and we outline the association between antiphospholipid antibodies and cardiac dysfunction.

Rapid cycling of lipid raft markers between the cell surface and Golgi complex.

The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 degrees C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.

Influence of human immunodeficiency virus type 1 envelope glycoprotein YXXL endocytosis/polarization signal on viral accessory protein functions.

INTRODUCTION: A tyrosine-based targeting signal in the intracytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is required for basolateral targeting of viral budding in polarized epithelial cells, concentration of viral assembly at one pole of infected lymphocytes, and rapid endocytosis of the glycoprotein from the plasma membrane. In HIV-1, the process of viral assembly and budding is complex and involves the participation of viral accessory proteins. STUDY DESIGN/METHODS: In this study, we examined whether the functions of the Nef, Vif, and Vpu proteins can influence or be influenced by the presence of envelope targeting signal in epithelial cells or lymphocytes. A series of proviral DNAs combining a mutation that inactivates the targeting signal with independent mutations in the three accessory proteins was constructed for this purpose. RESULTS: It was found that none of these three accessory proteins affected the basolateral release of the virus in polarized MDCK cells. Reciprocally, a mutation abolishing targeting of the viral envelope glycoprotein did not affect the phenotype conferred by the accessory proteins. Interestingly, the mutation abolishing targeting increased viral infectivity only in the presence of the Vpu protein. This phenotype was found to be associated with the release-enhancing function of Vpu and with an increased incorporation of viral envelope glycoprotein in virions. CONCLUSIONS: These data clearly show that accessory protein functions, and more specifically Vpu modulation of viral infectivity, can be affected by variations in the viral envelope glycoprotein.

Ethanol-glycine irrigating fluid for transurethral resection of the prostate in practice.

OBJECTIVE: To evaluate the usefulness of a tracer of 1% ethanol in 1. 5% glycine in the early detection of irrigation fluid absorption during transurethral resection of the prostate (TURP). PATIENTS AND METHODS: Patients (120) undergoing TURP were irrigated with 1% ethanol in 1.5% glycine solution and their expired air tested for alcohol every 10 min during the procedure. RESULTS: In all, 112 patients were assessed; over half of the patients absorbed the irrigation fluid and they had a significantly lower postoperative serum sodium concentration (P < 0.002). Fourteen patients (12.5%) absorbed over 500 mL and two (1.8%) developed clinical features of the TUR syndrome. The experience of the surgeon, the weight of resected chips and the operative duration were not significantly predictive of absorption. CONCLUSION: A tracer amount of ethanol in the irrigant is reliable for detecting absorption. Irrigating fluid absorption was unpredictable, thus supporting the case for routine monitoring.

An audit of the investigation and treatment of localised prostatic cancer in the south west region.

Prostate cancer constitutes a major health care dilemma in terms of treatment options available and increasing patient load on both a regional and national level. An audit was undertaken of all patients in the South West Region with localised prostate cancer newly diagnosed in 1993 to assess regional management of this disease. In 1993, 1407 patients were newly diagnosed as having prostatic cancer. Patients > 75 years old and those with a prostate-specific antigen (PSA) > 40 ng/ml were excluded, leaving 262 patients whose case notes were examined. The interval between referral and clinic (mean 67 days) was altered by the presence of a GP performed PSA, being shorter if the PSA was > 10 ng/ml (average 54 days) than if the PSA was < 10 ng/ml (average 104 days). Overall, 34% of patients underwent radical treatment (10% radical prostatectomy and 24% radiotherapy). In all, 27% received hormone manipulation or orchidectomy, and the remainder 'watchful waiting'. The majority (78%) of patients < 60 years old received radical treatment, as did 35% of those 60-70 years and 15% of 70-75 year olds. Over 90% of tumours were category T1 and were well or moderately differentiated. All patients had a histological diagnosis and 84% had their tumour staged before treatment. This study highlighted the need for improvements in patient assessment, improved note keeping and a regional cancer register to allow ongoing assessment of patient management. This audit of management of localised prostate cancer serves as a baseline from which to initiate and monitor improvements in the service regionally and will also allow assessment of the impact of such changes.

Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission.

Maturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.

MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes.

Human immunodeficiency virus (HIV) infection ultimately leads to the destruction of the CD4+ lymphocyte subset and the onset of AIDS. In recent years, several gene therapy procedures making use of retroviral vectors that selectively target HIV susceptible cells have been proposed in order to interfere with HIV productive infection. However, the HIV glycoproteins' inability to be incorporated in other heterologous retroviruses considerably limits true HIV cell tropism of such vectors. We now report the use of murine leukemia virus (MuLV) viral particles harboring a truncated form of the HIV glycoprotein for specific gene delivery. Reporter lacZ gene transfer was determined to be appropriately specific to CD4+ cells when HeLaCD4 cells or peripheral blood lymphocytes (PBLs) were infected with these pseudotyped MuLV virus vectors. In contrast, MuLV viruses harboring amphotropic MuLV envelope glycoproteins displayed a broad and nonspecific infection of PBL subpopulations. This new approach, taking advantage of the ability of truncated HIV envelope glycoproteins to be incorporated into heterologous retroviral particles, may foreseeably be used in future interventions based on the coordinated delivery of therapeutic gene products specifically to cell types susceptible to HIV infection.

Human immunodeficiency virus type 1 Vpr is a positive regulator of viral transcription and infectivity in primary human macrophages.

It is currently well established that HIV-1 Vpr augments viral replication in primary human macrophages. In its virion-associated form, Vpr has been suggested to aid efficient translocation of the proviral DNA into the cell nucleus. Although Vpr growth-arrests dividing T cells, the relevance of this biological activity in nondividing macrophages is unclear. Here we use Vpr-mutants to demonstrate that the molecular determinants involved in G2-arresting T cells are also involved in increasing viral transcription in macrophages, even though these cells are refractive to the diploid DNA status typical of G2 phase. Our results suggest that the two phenotypes, namely the nuclear localization and the G2-arrest activity of the protein, segregate functionally among the late and early functions of Vpr. The nuclear localization property of Vpr correlates with its ability to effectively target the proviral DNA to the cell nucleus early in the infection, whereas the G2-arrest phenotype correlates with its ability to activate viral transcription after establishment of an infection. These two functions may render Vpr's role essential and not accessory under infection conditions that closely mimic the in vivo situation, that is, primary cells being infected at low viral inputs.

Two distinct oncornaviruses harbor an intracytoplasmic tyrosine-based basolateral targeting signal in their viral envelope glycoprotein.

It has been clearly established that the budding of the human immunodeficiency virus (HIV-1), a lentivirus, occurs specifically through the basolateral membrane in polarized epithelial cells. More recently, the signal was assigned to a tyrosine-based motif located in the intracytoplasmic domain of the envelope glycoprotein, as previously observed on various other viral and cellular basolateral proteins. In the present study, expression of human T-cell leukemia virus type 1 (HTLV-1) or Moloney murine leukemia virus envelope glycoproteins was used for trans-complementation of an envelope-negative HIV-1. This demonstrated the potential of oncornaviral retrovirus envelope glycoproteins to confer polarized basolateral budding in epithelial Madin-Darby canine kidney cells (MDCK cells). Site-directed mutagenesis confirmed the importance of a common motif encompassing at least one crucial membrane-proximal intracytoplasmic tyrosine residue. The conservation of a similar basolateral maturation signal in different retroviruses further supports its importance in the biology of this group of viruses.

The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells.

Budding of retroviruses from polarized epithelial Madin-Darby canine kidney cells (MDCK) takes place specifically at the basolateral membrane surface. This sorting event is suspected to require a specific signal harbored by the viral envelope glycoprotein and it was previously shown that, as for most basolateral proteins, the intracytoplasmic domain plays a crucial role in this targeting phenomenon. It is well known that tyrosine-based motifs are a central element in basolateral targeting signals. In the present study, site-directed mutagenesis was used to generate conservative or non-conservative substitutions of each four intracytoplasmic tyrosines of the human immunodeficiency virus (HIV-1) envelope glycoprotein. This approach revealed that the membrane-proximal tyrosine is essential to ensure both the basolateral localization of envelope glycoprotein and the basolateral targeting of HIV-1 virions. Substitutions of the membrane-proximal tyrosine did not appear to affect incorporation of envelope glycoprotein into the virions, as assayed by virion infectivity and protein content, nor its capability to ensure its role in viral infection, as determined by viral multiplication kinetics. Altogether, these results indicate that the intracytoplasmic domain of the HIV-1 envelope glycoprotein harbors a unique, tyrosine-based, basolateral targeting signal. Such a tyrosine-based targeting signal may play a fundamental role in HIV transmission and pathogenesis.

Professional practice model research collaboration. Issues in longitudinal, multisite designs.

Implementing and evaluating professional practice models in dynamic healthcare settings is difficult and poses unique problems for nurse researchers and administrators. The authors describe issues associated with collaborating during the conduct of longitudinal studies.

The intracytoplasmic domain of gp41 mediates polarized budding of human immunodeficiency virus type 1 in MDCK cells.

Human immunodeficiency virus type 1 (HIV-1) has been shown to exhibit a specific basolateral release in polarized epithelial cells. Previous investigators have used vaccinia virus recombinants expressing HIV proteins to demonstrate that virus release is nonpolarized in the absence of viral envelope glycoproteins. In this study, we developed a transient expression system which allows the use of Madin-Darby canine kidney polarized epithelial cells directly grown on semipermeable membranes. This procedure allowed us to investigate polarized HIV viral budding following introduction of proviral DNA constructs. Expression of env gene products in trans demonstrated the ability to polarize env-negative viruses in a dose-dependent manner. The targeting signal for polarized virus release was shown to be present in the envelope gp41 transmembrane protein and absent from the gp120 portion of env. At least part of this signal is within the gp41 intracytoplasmic domain. Mutants of the p17gag matrix protein were shown to be nonpolarized only when unable to interact with the envelope glycoproteins. Together, these data are consistent with a model of polarized virus budding in which capsid proteins, lacking a targeting signal, are targeted for specific basolateral release via an interaction of p17 with the envelope glycoprotein containing the polarization signal in its intracytoplasmic domain.

Recurrent cerebral ischemia and mitral valve vegetation in a patient with antiphospholipid antibodies.

A 37-year-old woman presented with a history of acute loss of vision in her left eye and a history of recurrent transient ischemic attacks. Subsequent investigations revealed a prolonged PTT. The lupus anticoagulant and anticardiolipin antibodies (aCL) were identified in her serum. A cardiac murmur was heard and echocardiography demonstrated a mass on the mitral valve. Extensive studies for infection were negative. Cardioembolic phenomena were considered a possible cause of her cerebral ischemic events. The occurrence of nonbacterial endocardial verrucae is well described in systemic lupus erythematosus. The pathogenesis of this lesion remains speculative, however, its occurrence in our patient, with a lupus anticoagulant and aCL suggests a possible association. The clinical manifestations of thrombosis in patients with antiphospholipid antibodies are varied, and may include the development of thrombotic endocardial lesions.

Parathyroid venous sampling: anatomic considerations and results in 95 patients with primary hyperparathyroidism.

The most successful method of preoperative parathyroid adenoma localization is achieved by assay of venous samples for parathyroid hormone content. Selective sampling of the small veins draining the parathyroid glands increases the accuracy of prediction. Our review of 95 recent cases confirms an efficacy of prediction in 87.5%. The anatomy of the veins draining the parathyroid glands is outlined and we note variations that may occur in the inferior thyroid venous drainage. We consider contralateral venous flow to be of greater significance than has been recognized hitherto. This cross flow occurs through the thyroid plexus, and also via the vertebral venous plexus posteriorly and the anterior jugular veins in the front of the neck. Recent improvements in hormone assay combined with greater knowledge of the parathyroid venous drainage pattern of the individual patient may further improve the prediction rate.