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Background: Inflammatory bowel disease (IBD) is a lifelong digestive disease characterized by periods of severe inflammation and remission. To our knowledge, this is the first study showing a variable effect on ileitis severity from human gut microbiota isolated from IBD donors in remission and that of healthy controls in a mouse model of IBD., Methods: We conducted a series of single-donor intensive and nonintensive fecal microbiota transplantation (FMT) experiments using feces from IBD patients in remission and healthy non-IBD controls (N = 9 donors) in a mouse model of Crohn's disease (CD)-like ileitis that develops ileitis in germ-free (GF) conditions (SAMP1/YitFC; N = 96 mice)., Results: Engraftment studies demonstrated that the microbiome of IBD in remission could have variable effects on the ileum of CD-prone mice (pro-inflammatory, nonmodulatory, or anti-inflammatory), depending on the human donor. Fecal microbiota transplantation achieved a 95% ± 0.03 genus-level engraftment of human gut taxa in mice, as confirmed at the operational taxonomic unit level. In most donors, microbiome colonization abundance patterns remained consistent over 60 days. Microbiome-based metabolic predictions of GF mice with Crohn's or ileitic-mouse donor microbiota indicate that chronic amino/fatty acid (valine, leucine, isoleucine, histidine; linoleic; P < 1e-15) alterations (and not bacterial virulence markers; P > 0.37) precede severe ileitis in mice, supporting their potential use as predictors/biomarkers in human CD., Conclusion: The gut microbiome of IBD remission patients is not necessarily innocuous. Characterizing the inflammatory potential of each microbiota in IBD patients using mice may help identify the patients' best anti-inflammatory fecal sample for future use as an anti-inflammatory microbial autograft during disease flare-ups., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hypothesis: The high CMCs and low aggregation numbers of ionic micelles in the extreme electrolyte environment of ionic liquids (ILs) seem to be at odds with the effect of dilute aqueous electrolytes, which lower CMCs and promote elongated micelles. We hypothesise that the driving force for micellisation in ILs is determined by their underlying amphiphilic nanostructure, and that this can be controlled by mixing with water., Experiments: CMCs and micelle sizes of dodecyltrimethylammonium bromide (DTAB) are determined in mixed solvents comprising water and the ionic liquids ethylammonium nitrate (EAN), ethanolammonium nitrate (EtAN), and propylammonium nitrate (PAN) over a wide composition range. Their behaviour is compared with aqueous electrolytes up to their solubility limit. CMCs are determined by a variety of techniques, and their relative strengths critically evaluated. Micelle morphology is determined by small-angle neutron scattering., Findings: In water-rich mixtures, ILs do behave like simple electrolytes. Counterion binding dominates, both lowering the aqueous CMC and favouring a sphere-rod transition. However, even at modest concentrations, IL cations become incorporated into the micelle, causing the CMC to pass through a minimum, and arresting the sphere-rod transition. The efficiency of the cation depends on its amphiphilicity. As the IL content increases further, its role as a component of the bulk solvent becomes dominant: Only here does IL nanostructure influence micellization, as it increases alkyl chain solubility (EAN, PAN) and hence raises the CMC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tetanus is a vaccine-preventable disease that still commonly occurs in many low-income and middle-income countries, although it is rare in high-income countries. The disease is caused by the toxin of the bacterium Clostridium tetani and is characterised by muscle spasms and autonomic nervous system dysfunction. Global vaccination initiatives have had considerable success but they continue to face many challenges. Treatment for tetanus aims to control spasms and reduce cardiovascular instability, and consists of wound debridement, antitoxin, antibiotics, and supportive care. Recent research has focused on intravenous magnesium sulphate and intrathecal antitoxin administration as methods of spasm control that can avoid the need for ventilatory support. Nevertheless, without access to mechanical ventilation, mortality from tetanus remains high. Even with such care, patients require several weeks of hospitalisation and are vulnerable to secondary problems, such as hospital-acquired infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Neuropsychiatric disorders and type 2 diabetes (T2D) are major public health concerns proposed to be intimately connected. T2D is associated with increased risk of dementia, neuropsychiatric and mood disorders. Evidences of the involvement of insulin signaling on brain mechanisms related to depression indicate that insulin resistance, a hallmark of type 2 diabetes, could develop in the brains of depressive patients. In this article, we briefly review possible molecular mechanisms associating defective brain insulin signaling with reward system, neurogenesis, synaptic plasticity and hypothalamic-pituitary-adrenal (HPA) stress axis in depression. We further discuss the involvement of tumor necrosis factor α (TNFα) promoting defective insulin signaling and depressive-like behavior in rodent models. Finally, due to the high resistant rate of anti-depressants, novel insights into the link between insulin resistance and depression may advance the development of alternative treatments for this disease.

Despite long-standing availability of an effective vaccine, tetanus remains a significant problem in many countries. Outcome depends on access to mechanical ventilation and intensive care facilities and in settings where these are limited, mortality remains high. Administration of tetanus antitoxin by the intramuscular route is recommended treatment for tetanus, but as the tetanus toxin acts within the central nervous system, it has been suggested that intrathecal administration of antitoxin may be beneficial. Previous studies have indicated benefit, but with the exception of one small trial no blinded studies have been performed. The objective of this study is to establish whether the addition of intrathecal tetanus antitoxin reduces the need for mechanical ventilation in patients with tetanus. Secondary objectives: to determine whether the addition of intrathecal tetanus antitoxin reduces autonomic nervous system dysfunction and length of hospital/ intensive care unit stay; whether the addition of intrathecal tetanus antitoxin in the treatment of tetanus is safe and cost-effective; to provide data to inform recommendation of human rather than equine antitoxin. This study will enroll adult patients (≥16 years old) with tetanus admitted to the Hospital for Tropical Diseases, Ho Chi Minh City. The study is a 2x2 factorial blinded randomized controlled trial. Eligible patients will be randomized in a 1:1:1:1 manner to the four treatment arms (intrathecal treatment and human intramuscular treatment, intrathecal treatment and equine intramuscular treatment, sham procedure and human intramuscular treatment, sham procedure and equine intramuscular treatment). Primary outcome measure will be requirement for mechanical ventilation. Secondary outcome measures: duration of hospital/ intensive care unit stay, duration of mechanical ventilation, in-hospital and 240-day mortality and disability, new antibiotic prescription, incidence of ventilator associated pneumonia and autonomic nervous system dysfunction, total dose of benzodiazepines and pipecuronium, and incidence of adverse events. Trial registration: ClinicalTrials.gov NCT02999815 Registration date: 21 December 2016., Competing Interests: No competing interests were disclosed.

Tetanus remains a significant burden in many low- and middle-income countries. The tetanus toxin acts within the central nervous system and intrathecal antitoxin administration may be beneficial, but there are safety concerns, especially in resource-limited settings. We performed a pilot study to assess the safety and feasibility of intrathecal human tetanus immunoglobulin in five adults with tetanus before the conduct of a large randomized controlled trial. Intrathecal injection via lumbar puncture was given to all patients within a median 140 (range 100-165) minutes of intensive care unit (ICU) admission. There were no serious adverse effects associated with the procedure although three patients had probably related minor adverse events which resolved spontaneously. Median ICU length of stay was 14 (range 5-17) days. Two patients required mechanical ventilation and one developed a deep vein thrombosis. Within 240 days of hospital discharge, no patients died and all patients returned to work.

Sulfur-substituted nucleobases (i.e., thiobases) are a prospective class of compounds for clinical and cosmetic topical phototherapies. Recent investigations of several thiobases have revealed the ultrafast and efficient population of reactive triplet states upon ultraviolet-A (UVA) irradiation and the subsequent generation of singlet oxygen in high yield. In this contribution, we examine the photosensitizing activities of three of the most promising thiobase derivatives discovered to date: 2,4-dithiothymine, 2,4-dithiouracil, and 2,6-dithiopurine. These derivatives are shown to decrease the proliferation of human epidermoid carcinoma cells by up to 63 % in vitro, only upon activation with a low dose of UVA radiation (5 J cm -2 ). The generation of reactive oxygen species plays a minor role in the mode of action, suggesting these dithiobases may be effective within oxygen-deficient environments. Importantly, the photosensitized activity correlates with the magnitude of the triplet lifetime, which should guide the molecular design of next-generation photodynamic agents., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Epidemiological studies indicate that the use of artificial sweeteners doubles the risk for Crohn's disease (CD). Herein, we experimentally quantified the impact of 6-week supplementation with a commercial sweetener (Splenda; ingredients sucralose maltodextrin, 1:99, w/w) on both the severity of CD-like ileitis and the intestinal microbiome alterations using SAMP1/YitFc (SAMP) mice., Methods: Metagenomic shotgun DNA sequencing was first used to characterize the microbiome of ileitis-prone SAMP mice. Then, 16S rRNA microbiome sequencing, quantitative polymerase chain reaction, fluorescent in situ hybridization (FISH), bacterial culture, stereomicroscopy, histology, and myeloperoxidase (MPO) activity analyses were then implemented to compare the microbiome and ileitis phenotype in SAMP with that of control ileitis-free AKR/J mice after Splenda supplementation., Results: Metagenomics indicated that SAMP mice have a gut microbial phenotype rich in Bacteroidetes, and experiments showed that Helicobacteraceae did not have an exacerbating effect on ileitis. Splenda did not increase the severity of (stereomicroscopic/histological) ileitis; however, biochemically, ileal MPO activity was increased in SAMP treated with Splenda compared with nonsupplemented mice (P < 0.022) and healthy AKR mice. Splenda promoted dysbiosis with expansion of Proteobacteria in all mice, and E. coli overgrowth with increased bacterial infiltration into the ileal lamina propria of SAMP mice. FISH showed increase malX gene-carrying bacterial clusters in the ilea of supplemented SAMP (but not AKR) mice., Conclusions: Splenda promoted gut Proteobacteria, dysbiosis, and biochemical MPO reactivity in a spontaneous model of (Bacteroidetes-rich) ileal CD. Our results indicate that although Splenda may promote parallel microbiome alterations in CD-prone and healthy hosts, this did not result in elevated MPO levels in healthy mice, only CD-prone mice. The consumption of sucralose/maltodextrin-containing foods might exacerbate MPO intestinal reactivity only in individuals with a pro-inflammatory predisposition, such as CD.

Death receptor 3 (DR3), a member of the tumor necrosis factor receptor (TNFR) superfamily, has been implicated in regulating T-helper type-1 (T H 1), type-2 (T H 2), and type-17 (T H 17) responses as well as regulatory T cell (T reg ) and innate lymphoid cell (ILC) functions during immune-mediated diseases. However, the role of DR3 in controlling lymphocyte functions in inflammatory bowel disease (IBD) is not fully understood. Recent studies have shown that activation of DR3 signaling modulates T reg expansion suggesting that stimulation of DR3 represents a potential therapeutic target in human inflammatory diseases, including Crohn's disease (CD). In this study, we tested a specific DR3 agonistic antibody (4C12) in SAMP1/YitFc (SAMP) mice with CD-like ileitis. Interestingly, treatment with 4C12 prior to disease manifestation markedly worsened the severity of ileitis in SAMP mice despite an increase in FoxP3 + lymphocytes in mesenteric lymph node (MLN) and small-intestinal lamina propria (LP) cells. Disease exacerbation was dominated by overproduction of both T H 1 and T H 2 cytokines and associated with expansion of dysfunctional CD25 - FoxP3 + and ILC group 1 (ILC1) cells. These effects were accompanied by a reduction in CD25 + FoxP3 + and ILC group 3 (ILC3) cells. By comparison, genetic deletion of DR3 effectively reversed the inflammatory phenotype in SAMP mice by promoting the expansion of CD25 + FoxP3 + over CD25 - FoxP3 + cells and the production of IL-10 protein. Collectively, our data demonstrate that DR3 signaling modulates a multicellular network, encompassing T regs , T effectors, and ILCs, governing disease development and progression in SAMP mice with CD-like ileitis. Manipulating DR3 signaling toward the restoration of the balance between protective and inflammatory lymphocytes may represent a novel and targeted therapeutic modality for patients with CD.

Background: Ventilator-associated respiratory infection (VARI) is a significant problem in resource-restricted intensive care units (ICUs), but differences in casemix and etiology means VARI in resource-restricted ICUs may be different from that found in resource-rich units. Data from these settings are vital to plan preventative interventions and assess their cost-effectiveness, but few are available., Methods: We conducted a prospective observational study in four Vietnamese ICUs to assess the incidence and impact of VARI. Patients ≥ 16 years old and expected to be mechanically ventilated > 48 h were enrolled in the study and followed daily for 28 days following ICU admission., Results: Four hundred fifty eligible patients were enrolled over 24 months, and after exclusions, 374 patients' data were analyzed. A total of 92/374 cases of VARI (21.7/1000 ventilator days) were diagnosed; 37 (9.9%) of these met ventilator-associated pneumonia (VAP) criteria (8.7/1000 ventilator days). Patients with any VARI, VAP, or VARI without VAP experienced increased hospital and ICU stay, ICU cost, and antibiotic use ( p  < 0.01 for all). This was also true for all VARI ( p  < 0.01 for all) with/without tetanus. There was no increased risk of in-hospital death in patients with VARI compared to those without (VAP HR 1.58, 95% CI 0.75-3.33, p  = 0.23; VARI without VAP HR 0.40, 95% CI 0.14-1.17, p  = 0.09). In patients with positive endotracheal aspirate cultures, most VARI was caused by Gram-negative organisms; the most frequent were Acinetobacter baumannii (32/73, 43.8%) Klebsiella pneumoniae (26/73, 35.6%), and Pseudomonas aeruginosa (24/73, 32.9%). 40/68 (58.8%) patients with positive cultures for these had carbapenem-resistant isolates. Patients with carbapenem-resistant VARI had significantly greater ICU costs than patients with carbapenem-susceptible isolates (6053 USD (IQR 3806-7824) vs 3131 USD (IQR 2108-7551), p  = 0.04) and after correction for adequacy of initial antibiotics and APACHE II score, showed a trend towards increased risk of in-hospital death (HR 2.82, 95% CI 0.75-6.75, p  = 0.15)., Conclusions: VARI in a resource-restricted setting has limited impact on mortality, but shows significant association with increased patient costs, length of stay, and antibiotic use, particularly when caused by carbapenem-resistant bacteria. Evidence-based interventions to reduce VARI in these settings are urgently needed., Competing Interests: The study was approved by the Oxford Tropical Research Ethics Committee and local Ethics Committees of participating hospitals (Scientific and Ethics Committees of Bach Mai Hospital Hanoi, National Hospital for Tropical Diseases Hanoi, and Hospital for Tropical Diseases, Ho Chi Minh City). The study was carried out according to the principals of the Declaration of Helsinki and all participants gave written informed consent to take part in the study.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The human gut microbiome exerts a major impact on human health and disease, and therapeutic gut microbiota modulation is now a well-advocated strategy in the management of many diseases, including inflammatory bowel disease (IBD). Scientific and clinical evidence in support of complementary and alternative medicine, in targeting intestinal dysbiosis among patients with IBD, or other disorders, has increased dramatically over the past years. Delivery of "artificial" stool replacements for fecal microbiota transplantation (FMT) could provide an effective, safer alternative to that of human donor stool. Nevertheless, optimum timing of FMT administration in IBD remains unexplored, and future investigations are essential., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ultraviolet radiation (UVR) has a significant impact on human skin and is the major environmental factor for skin cancer formation. It is also believed that 80% of the signs of skin aging are attributed to UVR. UVR induces inflammatory changes in the skin via the increase in oxidative stress, DNA damage vascular permeability, and fluctuation in a myriad of cytokines. Acutely, UVR causes skin inflammation and DNA damage, which manifest as sunburn (erythema). ST266 is the secretome of proprietary amnion-derived cells that have been shown to reduce inflammation and accelerate healing of various wounds by promoting migration of keratinocytes and fibroblasts in preclinical animal studies. We hypothesized that ST266 has anti-inflammatory effects that can be used to reduce ultraviolet (UV) erythema and markers of inflammation. In this study, we examined the in vivo effects of ST266 on post UV-irradiated skin by measuring erythema, level of cyclobutane pyrimidine dimer (CPD), and expression level of xeroderma pigmentosum, complementation group A (XPA). We demonstrated that ST266 has the potential to reduce the acute effects of UV-induced skin damage when applied immediately after the initial exposure. In addition, ST266 is shown to reduce erythema, increase XPA DNA repair protein, and decrease damaged DNA., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

A 68-year-old gentleman presented with a lesion that resembled a pyogenic granuloma in his inferior fornix. The lesion was excised and biopsy demonstrated a proliferation of malignant spindle cells. Three weeks following initial excision, the lesion recurred and was removed via wedge excision of the eyelid. Definitive clearance was achieved through Mohs micrographic surgery. The patient received adjuvant postoperative radiotherapy and remains disease-free. This case demonstrates the need to consider sinister pathology in the setting of recurrent periocular lesions.

Fungi account for billions of infections worldwide. The second most prominent causative agent for fungal infections is Candida albicans (C. albicans). As strains of fungi become resistant to antifungal medications, new treatment modalities must be investigated to combat these infections. One approach is to employ photodynamic therapy (PDT). PDT utilizes a photosensitizer, light, and cellular O 2 to produce reactive oxygen species (ROS), which then induce oxidative stress resulting in apoptosis. Silicon phthalocyanine Pc 4 is a photosensitizer that has exhibited success in clinical trials for a myriad of skin diseases. The hydrophobic nature of Pc 4, however, poses significant formulation and delivery challenges in the use of this therapy. To mitigate these concerns, a drug delivery vehicle was synthesized to better formulate Pc 4 into a viable PDT agent for treating fungal infections. Utilizing poly(amidoamine) dendrimers as the framework for the vehicle, ∼13% of the amine chain ends were PEGylated to promote water solubility and deter nonspecific adsorption. In vitro studies with C. albicans demonstrate that the potency of Pc 4 was not hindered by the dendrimer vehicle. Encapsulated Pc 4 was able to effectively generate ROS and obliterate fungal pathogens upon photoactivation. The results presented within describe a nanoparticulate delivery vehicle for Pc 4 that readily kills drug-resistant C. albicans and eliminates solvent toxicity, thus, improving formulation characteristics for the hydrophobic photosensitizer.

The phase behavior and self-assembly structures of a series of amphiphilic diblock co-oligomers comprising an ionizable hydrophilic block (5 to 10 units of acrylic acid) and a hydrophobic block (5 to 20 units of n-butyl acrylate, t-butyl acrylate, or ethyl acrylate), synthesized by RAFT polymerization, have been examined by polarizing optical microscopy and small-angle X-ray scattering (SAXS). Self-assembled structure and lyotropic phase stability in these systems is highly responsive to the degree of ionization of the acrylic acid hydrophilic block (i.e., pH), concentration, and nature of the hydrophobic block. Increasing headgroup ionization switched the amphiphiles from behaving like soluble to insoluble surfactants. Liquid isotropic (micellar), hexagonal, lamellar, and discrete cubic phases were found under different solution conditions. The surfactant packing parameter was adapted to understand the self-assembly structures in these diblock co-oligomers. The hydrophobic chain structure and length were shown to strongly affect the relative stabilities of these phases, allowing the self-assembled structure to be varied at will.

The base pair d5SICS·dNaM was recently reported to incorporate and replicate in the DNA of a modified strain of Escherichia coli, thus making the world's first stable semisynthetic organism. This newly expanded genetic alphabet may allow organisms to store considerably more information in order to translate proteins with unprecedented enzymatic activities. Importantly, however, there is currently no knowledge of the photochemical properties of d5SICS or dNaM-properties that are central to the chemical integrity of cellular DNA. In this contribution, it is shown that excitation of d5SICS or dNaM with near-visible light leads to efficient trapping of population in the nucleoside's excited triplet state in high yield. Photoactivation of these long-lived, reactive states is shown to photosensitize cells, leading to the generation of reactive oxygen species and to a marked decrease in cell proliferation, thus warning scientists of the potential phototoxic side effects of expanding the genetic alphabet.

Exposure of the skin to ultraviolet (UV) irradiation causes many detrimental effects through mechanisms related to oxidative stress and DNA damage. Excessive oxidative stress can cause apoptosis and cellular dysfunction of epidermal cells leading to cellular senescence and connective tissue degradation. Direct and indirect damage to DNA predisposes the skin to cancer formation. Chronic UV exposure also leads to skin aging manifested as wrinkling, loss of skin tone, and decreased resilience. Fortunately, human skin has several natural mechanisms for combating UV-induced damage. The mechanisms operate on a diurnal rhythm, a cycle that repeats approximately every 24 hours. It is known that the circadian rhythm is involved in many skin physiologic processes, including water regulation and epidermal stem cell function. This study evaluated whether UV damage and the skin's natural mechanisms of inflammation and repair are also affected by circadian rhythm. We looked at UV-induced erythema on seven human subjects irradiated with simulated solar radiation in the morning (at 08:00 h) versus in the afternoon (at 16:00 h). Our data suggest that the same dose of UV radiation induces significantly more inflammation in the morning than in the afternoon. Changes in protein expression relevant to DNA damage, such as xeroderma pigmentosum, complementation group A (XPA), and cyclobutane pyrimidine dimers (CPD) from skin biopsies correlated with our clinical results. Both XPA and CPD levels were higher after the morning UV exposure compared with the afternoon exposure. , , J Drugs Dermatol. 2016;15(9):1124-1130.

The emergence of antibiotic-resistant strains in facultative anaerobic Gram-positive coccal bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), is a global health issue. Typically, MRSA strains are found associated with institutions like hospitals but recent data suggest that they are becoming more prevalent in community-acquired infections. It is thought that the incidence and prevalence of bacterial infections will continue to increase as (a) more frequent use of broad-spectrum antibiotics and immunosuppressive medications; (b) increased number of invasive medical procedures; and (c) higher incidence of neutropenia and HIV infections. Therefore, more optimal treatments, such as photodynamic therapy (PDT), are warranted. PDT requires the interaction of light, a photosensitizing agent, and molecular oxygen to induce cytotoxic effects. In this study, we investigated the efficacy and characterized the mechanism of cytotoxicity induced by photodynamic therapy sensitized by silicon phthalocyanine (Pc) 4 on (a) methicillin-sensitive Staphylococcus aureus (MSSA) (ATCC 25923); (b) community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) (ATCC 43300); and (c) hospital acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) (PFGE type 300). Our data include confocal image analysis, which confirmed that Pc 4 is taken up by all S. aureus strains, and viable cell recovery assay, which showed that concentrations as low as 1.0 μM Pc 4 incubated for 3 h at 37 °C followed by light at 2.0 J/cm2 can reduce cell survival by 2-5 logs. These results are encouraging, but before PDT can be utilized as an alternative treatment for eradicating resistant strains, we must first characterize the mechanism of cell death that Pc 4-based PDT employs in eliminating these pathogens.

Ventilator-associated pneumonia (VAP) is a serious healthcare-associated infection that affects up to 30 % of intubated and mechanically ventilated patients in intensive care units (ICUs) worldwide. The bacterial aetiology and corresponding antimicrobial susceptibility of VAP is highly variable, and can differ between countries, national provinces and even between different wards in the same hospital. We aimed to understand and document changes in the causative agents of VAP and their antimicrobial susceptibility profiles retrospectively over an 11 year period in a major infectious disease hospital in southern Vietnam. Our analysis outlined a significant shift from Pseudomonas aeruginosa to Acinetobacter spp. as the most prevalent bacteria isolated from quantitative tracheal aspirates in patients with VAP in this setting. Antimicrobial resistance was common across all bacterial species and we found a marked proportional annual increase in carbapenem-resistant Acinetobacter spp. over a 3 year period from 2008 (annual trend; odds ratio 1.656, P = 0.010). We further investigated the possible emergence of a carbapenem-resistant Acinetobacter baumannii clone by multiple-locus variable number tandem repeat analysis, finding a blaOXA-23-positive strain that was associated with an upsurge in the isolation of this pathogen. We additionally identified a single blaNDM-1-positive A. baumannii isolate. This work highlights the emergence of a carbapenem-resistant clone of A. baumannii and a worrying trend of antimicrobial resistance in the ICU of the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam., (© 2014 The Authors.)

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including age-related macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 μM Pc 4 followed by 670 to 675 nm light at 2.0 J/cm(2) reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A 31-year old man presented with swelling and purpura of the dorsum of the hands following sun exposure. There was a preceding lifelong history of photosensitivity, but this episode, which occurred after the recent commencement of oral iron therapy, and after recent alcohol ingestion, was much more severe than any preceding episode. Skin biopsy performed 48 h after the onset of symptoms showed features consistent with the early stages of leukocytoclastic vasculitis. Direct immunofluorescence showed homogeneous thick staining of the vessel walls with IgG, IgM and IgA, together with abundant perivascular fibrinogen. A subsequent periodic acid-Schiff (PAS) stain on the skin biopsy revealed thickening of the walls of dermal vessels, which was not discernible in routinely stained (hematoxylin/eosin) sections. The diagnosis of erythropoietic protoporphyria (EPP) was confirmed by significantly elevated erythrocyte protoporphyrin levels and positive plasma fluorimetry. The diagnosis of porphyria may have been missed by routine skin microscopy if not for the additional information provided by clinical history, direct immunofluorescence and PAS stain. The pathogenesis and histopathology of acute and chronic vascular changes in EPP are reviewed., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The fundamental mechanism of photodynamic therapy (PDT)-induced cell death has been characterized, but early critical PDT events in vivo remain incompletely defined. With the recent development in advanced fluorescence imaging modalities, such as intravital 2-photon laser scanning microscopy (2P-LSM), researchers are now able to investigate and visualize biological processes with high resolution in real time. This powerful imaging technology allows deep tissue visualization with single-cell resolution, thus providing dynamic information on the 3-dimensional architectural makeup of the tissue. The main goal of this study was to determine the cutaneous penetration of a topically applied photosensitizer, the silicon phthalocyanine Pc 4, into the skin of live animals and to assess the effective absorption of Pc 4 through the skin barrier. Our 2P-LSM images indicate that Pc 4 penetrates to the epidermal/dermal junction of mouse skin. The data also indicate that the degree of Pc 4 absorption is dose dependent. These findings represent initial steps that may help in improving the clinical utilization of topical Pc 4-PDT., (Published by Elsevier B.V.)

Mutations in α-actinin 4 (ACTN4) are linked to familial forms of focal segmental glomerulosclerosis (FSGS), a kidney disease characterized by proteinuria due to podocyte injury. The mechanisms underlying ACTN4 mutant-associated FSGS are not completely understood. Although α-actinins are better known to cross-link actin filaments and modulate cytoskeletal organization, we have previously shown that ACTN4 interacts with transcription factors including estrogen receptor and MEF2s and potentiates their transcriptional activity. Nuclear receptors including retinoic acid receptor (RAR) have been proposed to play a protective role in podocytes. We show here that ACTN4 interacts with and enhances transcriptional activation by RARα. In addition, FSGS-linked ACTN4 mutants not only mislocalized to the cytoplasm, but also lost their ability to associate with nuclear receptors. Consequently, FSGS-linked ACTN4 mutants failed to potentiate transcriptional activation by nuclear hormone receptors in podocytes. In addition, overexpression of these mutants suppressed the transcriptional activity mediated by endogenous wild-type ACTN4 possibly by a cytoplasmic sequestration mechanism. Our data provide the first link between FSGS-linked ACTN4 mutants and transcriptional activation by nuclear receptor such as RARα and peroxisome proliferator-activated receptor γ.

Healthcare-associated pneumonia (HCAP) is a common complication in patients with severe tetanus. Nursing tetanus patients in a semi-recumbent body position could reduce the incidence of HCAP. In a randomised controlled trial we compared the occurrence of HCAP in patients with severe tetanus nursed in a semi-recumbent (30°) or supine position. A total of 229 adults and children (aged ≥1 year) with severe tetanus admitted to hospital in Vietnam, were randomly assigned to a supine (n=112) or semi-recumbent (n=117) position. For patients maintaining their assigned positions and in hospital for>48h there was no significant difference between the two groups in the frequency of clinically suspected pneumonia [22/106 (20.8%) vs 26/104 (25.0%); p=0.464], pneumonia rate/1000 intensive care unit days (13.9 vs 14.6; p=0.48) and pneumonia rate/1000 ventilated days (39.2 vs 38.1; p=0.72). Mortality in the supine patients was 11/112 (9.8%) compared with 17/117 (14.5%) in the semi-recumbent patients (p=0.277). The overall complication rate [57/112 (50.9%) vs 76/117 (65.0%); p=0.03] and need for tracheostomy [51/112 (45.5%) vs 69/117 (58.9%); p=0.04) was greater in semi-recumbent patients. Semi-recumbent body positioning did not prevent the occurrence of HCAP in severe tetanus patients., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Neurons in the central amygdala (CeA) co-express dynorphin and corticotropin-releasing hormone (CRH). Moreover, the activity of both the CRH and dynorphin systems in CeA is altered by alcohol treatments, effects suggesting interactions between the CRH and dynorphin systems. Thus, the objectives of the present study were to investigate the effects of (1) activating CRH receptors (CRHRs) by microinjection of CRH in CeA and (2) blocking CRHRs by local microinjections of CRHR antagonists in the CeA on the alcohol-induced changes in the extracellular concentrations of dynorphin A1-8 with in vivo microdialysis experiments. Microdialysis probes with a microinjection port were implanted in the CeA of alcohol-naïve Sprague-Dawley rats. Microinjections of CRH or antalarmin, a CRH receptor type 1 (CRHR1) antagonist, or anti-sauvagine-30, a CRH receptor type 2 (CRHR2) antagonist, at the level of CeA were followed by an intraperitoneal injection of either saline or 2.8 g ethanol/kg body weight. The content of dynorphin A1-8 was determined in dialyzate samples obtained prior to and following the various treatments using a specific radioimmunoassay. Activation of CRHRs in CeA induced an increase in the extracellular concentrations of dynorphin A1-8. Moreover, acute alcohol administration increased the extracellular concentrations of dynorphin A1-8 in CeA, an effect that was attenuated by blocking CRHR2 with anti-sauvagine-30 microinjection but not blocking CRHR1 with antalarmin microinjection. Therefore, the findings suggest an interaction between the CRH and dynorphin A1-8 systems at the level of CeA in response to acute alcohol exposure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rationale: The endogenous opioid and corticotropin-releasing hormone (CRH) systems, present in the central amygdala (CeA), are implicated in alcohol consumption., Objectives: The purpose of this study is to investigate the hypothesis that, in CeA, alcohol stimulates CRH release, which then stimulates β-endorphin release., Materials and Methods: Rats were unilaterally implanted with a guide cannula to aim microdialysis probes in CeA. Experiment 1: rats received an intraperitoneal (IP) injection of various ethanol doses (0.0, 2.0, 2.4, or 2.8 g ethanol/kg body weight) and microdialysates were sampled at 30-min intervals to determine the effects over time of acute alcohol on the extracellular CRH concentrations in CeA. Experiment 2: phosphate-buffered saline, CRH, or CRH receptor (CRHR) antagonists (antalarmin or anti-sauvagine-30) was microinjected into CeA followed by a saline or 2.8 g/kg ethanol IP injection to determine the effects of CRHR activation or blockade in CeA on the basal and alcohol-stimulated release of β-endorphin. CRH and β-endorphin dialysate contents were determined using specific radioimmunoassays., Results: Acute alcohol induced a delayed increase in the extracellular CRH levels in CeA. Behavioural data showed no difference in locomotion between alcohol- and saline-treated rats. However, a transient increase in grooming was observed which did not correspond with alcohol-induced changes in CRH. Local CRH microinjections increased the extracellular β-endorphin concentrations in CeA. CRHR1 and CRHR2 blockade with microinjections of antalarmin and anti-sauvagine-30, respectively, attenuated the alcohol-induced increase of extracellular β-endorphin in CeA., Conclusions: Acute alcohol exerts indirect actions on CRH release and induced interactions of the CRH and β-endorphin systems in CeA.

Glucocorticoid hormones, including dexamethasone, induce apoptosis in lymphocytes and consequently are used clinically as chemotherapeutic agents in many hematologic malignancies. Dexamethasone also induces autophagy in lymphocytes, although the mechanism is not fully elucidated. Through gene expression analysis, we found that dexamethasone induces the expression of a gene encoding a stress response protein variously referred to as Dig2, RTP801, or REDD1. This protein is reported to inhibit mammalian target of rapamycin (mTOR) signaling. Because autophagy is one outcome of mTOR inhibition, we investigated the hypothesis that Dig2/RTP801/REDD1 elevation contributes to autophagy induction in dexamethasone-treated lymphocytes. In support of this hypothesis, RNAi-mediated suppression of Dig2/RTP801/REDD1 reduces mTOR inhibition and autophagy in glucocorticoid-treated lymphocytes. We observed similar results in Dig2/Rtp801/Redd1 knock-out murine thymocytes treated with dexamethasone. Dig2/RTP801/REDD1 knockdown also leads to increased levels of dexamethasone-induced cell death, suggesting that Dig2/RTP801/REDD1-mediated autophagy promotes cell survival. Collectively, these findings demonstrate for the first time that elevation of Dig2/RTP801/REDD1 contributes to the induction of autophagy.

Bcl-2 contributes to the pathophysiology and therapeutic resistance of chronic lymphocytic leukemia (CLL). Therefore, developing inhibitors of this protein based on a thorough understanding of its mechanism of action is an active and promising area of inquiry. One approach centers on agents (eg, ABT-737) that compete with proapoptotic members of the Bcl-2 protein family for binding in the hydrophobic groove formed by the BH1-BH3 domains of Bcl-2. Another region of Bcl-2, the BH4 domain, also contributes to the antiapoptotic activity of Bcl-2 by binding to the inositol 1,4,5-trisphosphate receptor (IP₃R) Ca²(+) channel, inhibiting IP(3)-dependent Ca²(+) release from the endoplasmic reticulum. We report that a novel synthetic peptide, modeled after the Bcl-2-interacting site on the IP₃R, binds to the BH4 domain of Bcl-2 and functions as a competitive inhibitor of the Bcl-2-IP₃R interaction. By disrupting the Bcl-2-IP₃R interaction, this peptide induces an IP₃R-dependent Ca²(+) elevation in lymphoma and leukemia cell lines and in primary CLL cells. The Ca²(+) elevation evoked by this peptide induces apoptosis in CLL cells, but not in normal peripheral blood lymphocytes, suggesting the involvement of the Bcl-2-IP₃R interaction in the molecular mechanism of CLL and indicating the potential merit of targeting this interaction therapeutically.

Class IIa HDACs including HDAC7 play a role in gene expression, cell differentiation, and animal development through their association with transcription factors such as myogenic enhancer factors 2 (MEF2s). In this study, we show that endogenous HDAC7 localizes to both the nucleus and the cytoplasm of C2C12 myoblasts but is exclusively retained in the cytoplasm of myotubes after completion of differentiation process. To elucidate the role of differential distribution of HDAC7 during myogenesis, we examined the effects of stably expressed HDAC7 mutants on myogenesis. Expression of nuclear-retained HDAC7 mutants significantly inhibits myogenesis in C2C12 cells and reduces the expression of muscle-specific myosin heavy chain (MHC) and myogenin. The inhibition in myocyte differentiation can be partially relieved by introduction of a mutation disrupting HDAC7:MEF2 interaction. Since phosphorylation of HDAC7 plays an important role in its nucleocytoplasmic shuttling, we further investigated the expression and distribution of phosphorylated HDAC7. To our surprise, the phosphorylation levels of HDAC7 at S344 and S479 were slightly decreased upon differentiation, whereas the phosphorylation of S178 was unchanged. Interestingly, a significant fraction of pS344- and/or pS479-HDAC7 localized to plasma membrane of myotubes. In addition, Ser178-phosphorylated (pS178) HDAC7 displays a predominantly actin filament-like structure before muscle differentiation. Consistent with this notion, HDAC7 partially colocalized with actin filaments; in particular, pS178-HDAC7 largely colocalized with actin filaments as indicated by phalloidin counter staining in myocytes. Furthermore, C2C12 cells expressing nuclear-retained HDAC7 display defects in migration. Our results provide novel insight into the mechanisms that regulate myocyte differentiation and migration by controlling the subcellular distribution of HDAC7 in differentiating myoblasts., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid(-/-) cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes., (© 2010 The Authors. Journal Compilation. The American Society of Photobiology.)

Our current focus on the effects of Photodynamic Therapy (PDT) using silicon phthalocyanine Pc 4 photosensitizer on malignant T lymphocytes arose due to preclinical observations that Jurkat cells, common surrogate for human T cell lymphoma, were more sensitive to Pc 4-PDT-induced killing than epidermoid carcinoma A431 cells. Mycosis fungoides (MF) as well as Sezary syndrome (SS) are variants of cutaneous T-cell lymphoma (CTCL) in which malignant T-cells invade the epidermis. In this study, we investigated the cytotoxicity of Pc 4-PDT in peripheral blood cells obtained from patients with SS and in skin biopsies of patients with MF. Our data suggest that Pc 4-PDT preferentially induces apoptosis of CD4(+)CD7(-) malignant T-lymphocytes in the blood relative to CD11b(+) monocytes and nonmalignant T-cells. In vivo Pc 4-PDT of MF skin also photodamages the antiapoptotic protein Bcl-2.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Rationale: There is experimental evidence that indicates that the endogenous opioid system of the central nucleus of the amygdala (CeA) may mediate some of the reinforcing effects of ethanol. However, the precise interactions of ethanol with the endogenous opioid system at the level of the CeA have not been investigated., Objectives: The aim of the current study was to investigate the hypothesis that acute systemic ethanol administration will increase the release of endogenous opioid peptides at the level of the CeA in a time- and dose-dependent manner., Materials and Methods: Rats were implanted with a unilateral guide cannula to aim microdialysis probes at the CeA. Intraperitoneal injections of saline and various doses of ethanol (0.8, 1.6, 2.0, 2.4, and 2.8 g ethanol/kg body weight) were administered to the rats. Dialysate samples were collected at 30-min intervals at distinct time points prior to and following treatment. Radioimmunoassays specific for beta-endorphin, met-enkephalin, and dynorphin A1-8 were used to determine the effect of ethanol on the content of the opioid peptides in the dialysate., Results: We report that the 2.8-g/kg dose of ethanol induced a long-lasting increase in beta-endorphin release from 60 min onwards following administration and, later, an ongoing increase in dynorphin A1-8 release. None of the ethanol doses tested elicited significant changes in dialysate met-enkephalin content compared to the saline treatment., Conclusions: Acute systemic ethanol administration induced a dose- and time-dependent increase in beta-endorphin and dynorphin A1-8 release at the level of the CeA, which may be involved in ethanol consumption.

The transcriptional modulator Cited2 is induced by various biological stimuli including hypoxia, cytokines, growth factors, lipopolysaccharide (LPS) and flow shear. In this study, we report that Cited2 is required for mouse fetal liver development. Cited2(-/-) fetal liver displays hypoplasia with higher incidence of cell apoptosis, and exhibits disrupted cell-cell contact, disorganized sinusoidal architecture, as well as impaired lipid metabolism and hepatic gluconeogenesis. Furthermore, we demonstrated the physical and functional interaction of Cited2 with liver-enriched transcription factor HNF4alpha. Chromatin immunoprecipitation (ChIP) assays further confirmed the recruitment of Cited2 onto the HNF4alpha-responsive promoters and the reduced HNF4alpha binding to its target gene promoters in the absence of Cited2. Taken together, this study suggests that fetal liver defects in mice lacking Cited2 result, at least in part, from its defective coactivation function for HNF4alpha.

The aberrant association of promyelocytic leukemia protein-retinoic acid receptor-alpha (PML-RARalpha) with corepressor complexes is generally thought to contribute to the ability of PML-RARalpha to regulate transcription. We report here that PML-RARalpha acquires aberrant association with coactivators. We show that endogenous PML-RARalpha interacts with the histone acetyltransferases CBP, p300, and SRC-1 in a hormoneindependent manner, an association not seen for RARalpha. This hormone-independent coactivator binding activity requires an intact ligand-binding domain and the NR box of the coactivators. Confocal microscopy studies demonstrate that exogenous PML-RARalpha sequesters and colocalizes with coactivators. These observations correlate with the ability of PML-RARalpha to attenuate the transcription activation of the Notch signaling downstream effector, CBF1, and of the glucocorticoid receptor. This includes attenuation of the glucocorticoid-induced leucine zipper (GILZ) and FLJ25390 target genes of the endogenous glucocorticoid receptor. Furthermore, treatment of NB4 cells with all-trans-retinoic acid, which promotes PML-RARalpha degradation, resulted in increased activation of GILZ. On the basis of these findings, we propose a model in which the hormone-independent association between PML-RARalpha and coactivators contributes to its ability to regulate gene expression.

Histone deacetylase 7 (HDAC7) is a member of class IIa HDACs that regulate myocyte enhancer factor-2 (MEF2)-mediated transcription and participate in multiple cellular processes such as T cell apoptosis. We have identified alpha-actinin 1 and 4 as class IIa HDAC-interacting proteins. The interaction domains are mapped to C terminus of alpha-actinin 4 and amino acids 72-172 of HDAC7. A point mutation in HDAC7 that disrupts its association with MEF2A also disrupts its association with alpha-actinin 4, indicating that MEF2A and alpha-actinin 4 binding sites largely overlap. We have also isolated a novel splice variant of alpha-actinin 4 that is predominantly localized in the nucleus, a pattern distinct from the full-length alpha-actinin 4, which is primarily distributed in the cytoplasm and plasma membrane. Using small interfering RNA, chromatin immunoprecipitation, and transient transfection assays, we show that alpha-actinin 4 potentiates expression of TAF55, a putative MEF2 target gene. Loss of MEF2A interaction correlates with loss of the ability of alpha-actinin 4 to potentiate TAF55 promoter activity. Ectopic expression of alpha-actinin 4, but not the mutant defective in MEF2A association, leads to disruption of HDAC7.MEF2A association and enhancement of MEF2-mediated transcription. Taken together, we have identified a novel mechanism by which HDAC7 activity is negatively regulated and uncovered a previously unknown function of alpha-actinin 4.

Background: Pharmacological studies have implicated the endogenous opioid system in mediating alcohol intake. Other evidence has shown that alcohol administration can influence endorphinergic and enkephalinergic activity, while very few studies have examined its effect on dynorphinergic systems. The aim of the present study was to investigate the effect of alcohol administration or a mechanical stressor on extracellular levels of dynorphin A(1-8) in the rat nucleus accumbens-a brain region that plays a significant role in the processes underlying reinforcement and stress., Methods: Male Sprague-Dawley rats were implanted with a microdialysis probe aimed at the shell region of the nucleus accumbens. Artificial cerebrospinal fluid was pumped at a rate of 1.5 microL/min in awake and freely moving animals and the dialysate was collected at 30-minute intervals. In one experiment, following a baseline period, rats were injected intraperitoneally with either physiological saline or 1 of 3 doses of alcohol, 0.8, 1.6, or 3.2 g ethanol/kg body weight. In a second experiment, following a baseline period, rats were applied a clothespin to the base of their tail for 20 minutes. The levels of dynorphin A(1-8) in the dialysate were analyzed with solid-phase radioimmunoassay., Results: Relative to saline-treated controls, an alcohol dose of 1.6 and 3.2 g/kg caused a transient increase in the extracellular levels of dynorphin A(1-8) in the first 30 minutes of alcohol administration. However, the effect resulting from the high 3.2 g/kg dose was far more pronounced and more significant than with the moderate dose. There was no effect of tail pinch on dynorphin A(1-8) levels in the nucleus accumbens., Conclusions: In this experiment, a very high dose of alcohol was especially capable of stimulating dynorphin A(1-8) release in the nucleus accumbens. Dynorphin release in the accumbens has been previously associated with aversive stimuli and may thus reflect a system underlying the aversive properties of high-dose alcohol administration. However, the lack of effect of tail-pinch stress in the present study suggests that dynorphin A(1-8) is not released in response to all forms of stressful/aversive stimuli.

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).

Rationale: Guanfacine is an alpha2-adrenergic receptor agonist that has been shown to have beneficial effects on working memory and attentional functions in monkeys and in patients with attention deficit hyperactivity disorder., Objectives: The aim of this study was to further investigate the cognitive-enhancing properties of guanfacine using an established battery of tasks measuring executive and memory functions., Methods: Sixty healthy male volunteers were randomised into three groups. Cognitive testing was performed from +2 to +4 h after double-blind administration of a single oral dose of 1 or 2 mg of guanfacine or placebo., Results: Systolic blood pressure was significantly reduced by both doses of guanfacine at the end of the testing session. There were no statistically significant effects on any of the cognitive measures. Two trend effects were observed with poorer performance on digit span backward and slower 'Go' reaction times after guanfacine., Conclusion: This study found no improvement of prefrontal memory or executive functions after guanfacine. Negative effects on blood pressure and trend effects on digit span backward and go reaction time indicate a mild sedative effect of guanfacine at these doses, possibly via mechanisms of autoreceptor down-regulation.

Phthalocyanine (Pc) 4, like many photosensitizers for photodynamic therapy (PDT), localizes to intracellular membranes, especially mitochondria. Pc 4-PDT photodamages Bcl-2 and Bcl-xL, antiapoptotic proteins interacting with the permeability transition pore complex that forms at contact sites between the inner and outer mitochondrial membranes. These complexes and the inner membrane are unique in containing the phospholipid cardiolipin. Nonyl-acridine orange (NAO) is a specific probe of cardiolipin. Here we show evidence for fluorescence resonance energy transfer from NAO to Pc 4, defining a binding site for the photosensitizer. This observation establishes an innovative tool for exploring the localization of other photosensitizers and additional fluorescent, mitochondrion-localizing drugs having appropriate spectral properties.

Photodynamic therapy using the photosensitizer Pc 4 and red light photochemically destroys the antiapoptotic protein Bcl-2 and induces apoptosis. To characterize the requirements for photodamage, we transiently transfected epitope-tagged Bcl-2 deletion mutants into DU-145 cells. Using confocal microscopy and Western blots, wild-type Bcl-2 and mutants with deletions near the N terminus were found in mitochondria, endoplasmic reticulum, and nuclear membranes and were photodamaged. A mutant missing the C terminus, including the transmembrane domain, spread diffusely in cells and was not photodamaged. Bcl-2 missing alpha-helices 5/6 was also not photodamaged. Bcl-2 missing only one of those alpha-helices, with or without substitutions of the singlet oxygen-targeted amino acids, behaved like wild-type Bcl-2 with respect to localization and photodamage. Using green fluorescent protein (GFP)-tagged Bcl-2 or mutants in live cells, no change in either the localization or the intensity of GFP fluorescence was observed in response to Pc 4 photodynamic therapy. Western blot analysis of either GFP- or Xpress-tagged Bcl-2 revealed that the photodynamic therapy-induced disappearance of the Bcl-2 band was accompanied by the appearance of bands indicative of heavily cross-linked Bcl-2 protein. Therefore, the alpha(5)/alpha(6) region of Bcl-2 is required for photodamage and cross-linking, and domain-dependent photodamage to Bcl-2 offers a unique mechanism for activation of apoptosis.