Parameter estimation using weighted total least squares in the two-compartment exchange model.
The linear least squares (LLS) estimator provides a fast approach to parameter estimation in the linearized two-compartment exchange model. However, the LLS method may introduce a bias through correlated noise in the system matrix of the model. The purpose of this work is to present a new estimator for the linearized two-compartment exchange model that takes this noise into account. To account for the noise in the system matrix, we developed an estimator based on the weighted total least squares (WTLS) method. Using simulations, the proposed WTLS estimator was compared, in terms of accuracy and precision, to an LLS estimator and a nonlinear least squares (NLLS) estimator. The WTLS method improved the accuracy compared to the LLS method to levels comparable to the NLLS method. This improvement was at the expense of increased computational time; however, the WTLS was still faster than the NLLS method. At high signal-to-noise ratio all methods provided similar precisions while inconclusive results were observed at low signal-to-noise ratio. The proposed method provides improvements in accuracy compared to the LLS method, however, at an increased computational cost. Magn Reson Med 79:561-567, 2017. © 2017 International Society for Magnetic Resonance in Medicine.


Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells.
Activity-based monitoring of cell-secreted proteases has gained significant interest due to the implication of these substances in diverse cellular functions. Here, we demonstrated a cell-based method of monitoring protease activity using fluorescent cell-permeable peptides. The activatable peptide consists of anionic (EEEE), cleavable, and cationic sequences (RRRR) that enable intracellular delivery by matrix metalloproteinase-2 (MMP2), which is secreted by living cancer cells. Compared to HT-29 cells (MMP2-negative), HT-1080 cells (MMP2-positive) showed a strong fluorescence response to the short fluorescent peptide via cell-secreted protease activation. Our approach is expected to find applications for the rapid visualization of protease activity in living cells.


Mice lacking melanin-concentrating hormone are hypophagic and lean.
Feeding is influenced by hypothalamic neuropeptides that promote (orexigenic peptides) or inhibit feeding. Of these, neuropeptide Y (NPY) in the arcuate nucleus and melanin-concentrating hormone (MCH) and orexins/hypocretins in the lateral hypothalamus have received attention because their expression is increased during fasting and because they promote feeding when administered centrally. Surprisingly, absence of the orexigenic neuropeptide NPY fails to alter feeding or body weight in normal mice. As deficiency of a single component of the pathway that limits food intake (such as leptin or receptors for melanocortin-4) causes obesity, it has been suggested that orexigenic signals are more redundant than those limiting food intake. To define further the physiological role of MCH and to test the redundancy of orexigenic signals, we generated mice carrying a targeted deletion of the MCH gene. MCH-deficient mice have reduced body weight and leanness due to hypophagia (reduced feeding) and an inappropriately increased metabolic rate, despite their reduced amounts of both leptin and arcuate nucleus pro-opiomelanocortin messenger RNA. Our results show that MCH is a critical regulator of feeding and energy balance which acts downstream of leptin and the melanocortin system, and that deletion of a gene encoding a single orexigenic peptide can result in leanness.


Addressing Diversity, Equity, and Inclusivity Contributions in Academic Review.
The nursing profession and academic environments are increasingly calling for nurse educators to incorporate diversity, equity, and inclusion (DEI) goals and actions into their professional practice. Such work aligns with institutional and professional missions of social justice and antiracism. Historically, there has been an undervaluing and lack of recognition in academia of DEI contributions and qualitative or community-based research focused on health equity. This lack of recognition may disproportionately impact faculty who focus their scholarship and research on marginalized communities. The current state of the academic review of DEI contributions for appointment, promotion, and tenure is presented. Examples for teaching, research and scholarship, practice, and service are provided. Recommendations include expanding recognition and evaluation of academic scholarship and advocacy against efforts to dismantle social justice and antiracism advances within academia. Examining hegemonic norms is vital to promote more equitable power structures in academic environments.


Side effects of danazol compared with an ethinyloestradiol/norgestrel combination when used for postcoital contraception.
A postcoital contraceptive with a lower incidence of nausea and vomiting than oestrogen-progestogen combinations would be a significant advance. During a nine-month period, 101 women were treated at the Margaret Pyke Centre in London with either an oestrogen-progestogen combination or with danazol. A comparison of the side effects of each drug is reported. Those treated with danazol were six times less likely to experience nausea and none vomited. With the exception of breast symptoms, other side effects were five times less common in women receiving danazol. These differences give danazol a clear advantage in terms of patient acceptability. Further experience will enable the efficacy of danazol to be evaluated and so determine whether this drug should become the preferred hormonal postcoital treatment.


Tetramine dichloro-palladium subcellular localization in the kidney: electron microprobe study.
Palladium salt has been used for some time in experimental therapy protocols; with this in mind, we carried out a study of the effect of tetramine dichloro-palladium (soluble salt) upon kidney cells. Using an electron microprobe, we were able to detect the presence of palladium associated with sulfur and iron in the lysosomes of the proximal tubule cells. Our results were compared with those obtained using Cis-diaminedichloro-platinum (Cis-DDP), an anti-cancer drug used in the treatment of diverse tumors. The mechanism of intralysosomal concentration of palladium as a non soluble salt associated with sulfur appeared to be related to local sulfatase activity. Finally, iron concentration appeared to be related to the inhibition process of erythropoiesis.


Responsiveness of prehaemolytic copper poisoning in sheep from a specific pathogen-free environment to a relatively high dose of tetrathiomolybdate.
Efficacy of ammonium tetrathiomolybdate (3.4 mg/kg LW, TTM(3.4)) was monitored in nine, specific pathogen-free sheep with mild-to-severe, prehaemolytic copper poisoning (pre-HCP). Five sheep were given three subcutaneous injections over seven days and four began a shorter, five-day course four days later. Plasma bile acid (BA) and glutamate dehydrogenase (GDH) had fallen significantly after six days but BA briefly rose again between days 10 to 18. Aspartate aminotransferase (AST), δ-glutamyl transferase (GGT) were later to decline but presented little evidence of hepatotoxicity by day 45. Erythrocyte superoxide dismutase (ESOD) was rapidly inhibited, activities falling by 65 per cent within three days and taking 25 days to recover. Trichloroacetic (TCA)-insoluble copper increased by 6 to 8 μmol/l after three days but had largely disappeared by day 18. Six lambs had become hypercupraemic by day 18 due to a rise in TCA-soluble Cu. ESOD activity fell again by 188 ± 29.5 U/gHb between days 25 and 45 but had largely recovered by day 62. TTM(3.4) gave better control of hepatotoxicity than TTM(1.7) had done in more severely affects cohorts but at the expense of greater inhibition of ESOD. Treatment of pre-HCuP should rely more on reducing copper absorption until more is known of the side effects of TTM on cuproenzymes.


Cadherin-7 regulates mossy fiber connectivity in the cerebellum.
To establish highly precise patterns of neural connectivity, developing axons must stop growing at their appropriate destinations and specifically synapse with target cells. However, the molecular mechanisms governing these sequential steps remain poorly understood. Here, we demonstrate that cadherin-7 (Cdh7) plays a dual role in axonal growth termination and specific synapse formation during the development of the cerebellar mossy fiber circuit. Cdh7 is expressed in mossy fiber pontine nucleus (PN) neurons and their target cerebellar granule neurons during synaptogenesis and selectively mediates synapse formation between those neurons. Additionally, Cdh7 presented by mature granule neurons diminishes the growth potential of PN axons. Furthermore, knockdown of Cdh7 in PN neurons in vivo severely impairs the connectivity of PN axons in the developing cerebellum. These findings reveal a mechanism by which a single bifunctional cell-surface receptor orchestrates precise wiring by regulating axonal growth potential and synaptic specificity.


Portuguese recommendations for the treatment of atopic dermatitis with biologic therapy and JAK inhibitors in adult patients.
Atopic dermatitis is a highly prevalent chronic, immune-mediated inflammatory skin disease with a significant burden on patients, families and healthcare systems. This article presents recommendations developed by the Atopic Dermatitis Group of the Portuguese Society of Dermatology and Venereology addressing several clinical questions that arise in the management and care of moderate-to-severe atopic dermatitis with biologic agents and Janus kinase (JAK) inhibitors based on the available evidence. The recommendations were generated after a thorough evaluation of existing guidelines on the treatment of atopic dermatitis, publications concerning new biologics and JAK inhibitors not yet incorporated into existing guidelines, and expert-based recommendations. It also includes considerations on atopic dermatitis severity, indications for initiating biologic agents and JAK inhibitors, parameters to be considered in the treatment choice, in particular treatment goals, and recommendations for the use, screening and monitoring of these therapies.


The association between the signalment, common causes of canine otitis externa and pathogens.
To determine whether associations exist between pathogens, allergies, conformational abnormalities, endocrinopathies and signalment in canine otitis externa (OE). Medical records of 149 dogs which met predetermined inclusion criteria were evaluated retrospectively. Correlations between pathogens and the presence of allergy, endocrinopathy, conformational abnormalities and signalment were evaluated statistically. The shar-pei, German shepherd and cocker spaniel breeds were over-represented compared with the hospital's breed distribution (P<0·001). German shepherd dogs and cocker spaniels were statistically more prone to infection with rod-shaped organisms and Labrador retrievers less than other breeds (P=0·034). Almost all dogs that were older than five years when diagnosed with OE had cocci (P=0·01) and also had higher levels of rods (P=0·028). The incidence of rods was higher in endocrinopathies (P=0·004), while that of Malassezia spp. tended to be higher in allergies (P=0·098). There were no statistically significant differences among the groups for all the other parameters examined. OE infection is usually not influenced by primary causes or predisposing factors. Endocrinopathies may be followed by a more severe otitis, however. OE may be more severe when it affects older dogs.


Ultrasound image of healthy skin in newborns in the first 24 hours of life.
Ultrasound imaging is a safe, repeatable and easily available imaging procedure. Based on these qualities, it may become a useful tool for skin assessment in newborns. Aims: The aim of the study was to evaluate the usefulness of high-frequency ultrasound imaging for neonatal skin assessment. Another aim was to identify differences in ultrasound features of the skin in newborns depending on the examination site, sex, age, birth weight, and arterial blood gas results. A total of 72 newborns in the first 24 hours of life, without any skin lesions, were included in the study. All newborns underwent ultrasound skin examinations in three body sites (forearm, abdomen and thigh) during the first 24 hours of life. DermaMed Ultrasound Scanner with a 48 MHz probe was used for imaging. A total of three structures were identified in the ultrasound images: epidermal echo, dermis, and subcutaneous tissue. The study assessed the thickness of the epidermis and dermis, and the echogenicity of the dermis and subcutaneous tissue. Data were analysed to determine possible links with sex, post-conceptional age, body weight, birth route, and results of umbilical cord blood gas analysis. Depending on the body site examined, the mean epidermal thickness was 0.081 to 0.083 mm, while the mean thickness of the dermis ranged between 0.679 and 0.722 mm. The newborns with higher birth weights were shown to have a thicker epidermis regardless of the examined site [R (correlation coefficient) for the forearm: 0.47 (p <0.001), abdomen: 0.53 (p <0.001), thigh: 0.48 (p <0.001)]. A positive correlation was found between epidermal and dermal thickness (R = 0.34; p = 0.004), but a comparison of the three examined sites revealed no significant differences in the thickness of the two structures. The sex of the newborn had no significant effect on the ultrasound features of the skin. None of the ultrasound parameters under study was found to correlate with the pH level in umbilical cord blood gas analysis. Subcutaneous oedema was detected in the examined sites in all the newborns studied. High-frequency ultrasound imaging may become a useful method for neonatal skin assessment, complementing existing diagnostic techniques for monitoring pathologically altered skin.


Intermittent pneumatic compression in immobile patients.
The purpose of this study is to stress the value of using intermittent pneumatic compression (IPC) in immobile patients. The use of IPC helps prevent limb oedema and the associated skin changes frequently seen on the legs of the immobile patient. Oedema formation is caused by an increase of fluid extravasation, while skin changes including leg ulcers are mainly because of a deficiency of the venous and lymphatic pumps. Conventional compression stockings and bandages impede leg swelling but are less efficient in supporting the deficient veno-lymphatic pump when patients are unable to move. In this situation, actively compressing the limb using IPC is a very meaningful and effective treatment option. Because of a lack of literature on the specific indication of IPC in immobile patients, experimental studies and randomised controlled trials in similar situations are reviewed. IPC is a very effective although underused treatment modality, especially in immobile, wheelchair-bound patients. By inflation and deflation of the air-filled garments, IPC produces cycles of pressure waves on the leg, thus mimicking the working and resting pressures applied by compression bandages. IPC not only reduces leg swelling but also augments the veno-lymphatic pump, which is essential for the restoration of the damaged microcirculation of the skin.


[Hematological data analysis in children with thalassemia trait or hemoglobin H disease in Taiwan].
In the past several years, we have collected 91 cases diagnosed as beta-thalassemia trait, 94 cases as alpha-thalassemia trait and 43 cases as hemoglobin H disease. The ages ranged from 6 months to 15 years. The hematological data were analyzed. We calculated the mean +/- SD for each item of the erythrocyte indices (RBC, Hgb, Hct, MCV, MCH, MCHC, +/- RDW) and the Hb electrophoretic results (Hb A, Hb A2, Hb F, Hb H & Bart's) in each group, and tabulated them. Then, comparisons were made between beta and alpha-thalassemia traits or between the thalassemia trait and hemoglobin H disease. We found that each of the mean values for Hgb, Hct, MCV and MCH for children with the beta-thalassemia trait was significantly lower than that for children with the alpha-thalassemia trait. The mean MCV value of the beta-thalassemia trait group was 60.7 +/- 3.4 fl, and 87% of the MCV values in this group ranged from 55 to 65 fl. The mean MCV value for the alpha-thalassemia trait group was 63.3 +/- 3.3 fl, and 56% of the MCV values in this group ranged from 60-65 fl. The MCV values for all of the 185 children studied with either the beta- or alpha-thalassemia trait ranged from 54 to 71 fl, and were far below the normal ranges for MCV values for each age-specific group. The mean percentage of deviation of MCV from normal was greater than that of Hgb in both the beta- and alpha-thalassemia trait groups. A comparison of those with beta-thalassemia trait 6 years old and under and those over 6 years showed that both the mean values of RBC and Hgb were higher in the older subgroup, while the mean MCV values did not show any significant differences between these 2 subgroups.(ABSTRACT TRUNCATED AT 250 WORDS)


SCIP: a single-cell image processor toolbox.
Each cell is a phenotypically unique individual that is influenced by internal and external processes, operating in parallel. To characterize the dynamics of cellular processes one needs to observe many individual cells from multiple points of view and over time, so as to identify commonalities and variability. With this aim, we engineered a software, 'SCIP', to analyze multi-modal, multi-process, time-lapse microscopy morphological and functional images. SCIP is capable of automatic and/or manually corrected segmentation of cells and lineages, automatic alignment of different microscopy channels, as well as detect, count and characterize fluorescent spots (such as RNA tagged by MS2-GFP), nucleoids, Z rings, Min system, inclusion bodies, undefined structures, etc. The results can be exported into *mat files and all results can be jointly analyzed, to allow studying not only each feature and process individually, but also find potential relationships. While we exemplify its use on Escherichia coli, many of its functionalities are expected to be of use in analyzing other prokaryotes and eukaryotic cells as well. We expect SCIP to facilitate the finding of relationships between cellular processes, from small-scale (e.g. gene expression) to large-scale (e.g. cell division), in single cells and cell lineages. http://www.ca3-uninova.org/project_scip. Supplementary data are available at Bioinformatics online.


Brown seaweed- (Tasco) treated conserved forage enhances antioxidant status and immune function in heat-stressed wether lambs.
Twenty-seven wether lambs were utilized to evaluate select innate immunity and oxidative stress in response to diet and heat stress. Dietary treatments were: (i) control (tall fescue) hay = no Tasco (tradename for the extract of the brown seaweed, Ascophyllum nodosum, Acadian Sealants Ltd, Nova Scotia, Canada); (ii) pre-harvest Tasco-Forage-treated hay and (iii) control hay + post-harvest Tasco-EX. Tasco-Forage and Tasco-EX are two forms of the Tasco extract that are either applied to foliage or used for direct feeding, respectively. All lambs were supplemented with soyabean meal and trace mineralized salt. Heat stress was applied for 10 days with measurements obtained at days 0, 4 and 10. A heat x treatment interaction indicated hay with Tasco enhanced monocyte oxidative burst through short duration (p < 0.05) and long duration (p < 0.10) heat stress. Phagocytic activity was influenced by days of heat stress (p < 0.001) and treatment (p = 0.02) with post-harvest Tasco lambs exhibiting the greatest immune enhancement (p < 0.05). Red and white blood cell glutathione peroxidase increased by heat stress day 10 in Tasco lambs. Superoxide dismutase activity was increased and lipid hydroperoxide metabolites minimized (p < 0.01) through long duration heat stress in the pre-harvest Tasco group. Tasco treatment of tall fescue hay prior to harvest appears to provide residual effects on animal antioxidant availability in short-duration heat stress. Tasco supplementation to post-harvest fescue hay enhances immune function and protects against prolonged heat-induced oxidative stress.


Early life vincristine exposure evokes mechanical pain hypersensitivity in the developing rat.
Vincristine (VNC) is commonly used to treat pediatric cancers, including the most prevalent childhood malignancy, acute lymphoblastic leukemia. Although clinical evidence suggests that VNC causes peripheral neuropathy in children, the degree to which pediatric chemotherapeutic regimens influence pain sensitivity throughout life remains unclear, in part because of the lack of an established animal model of chemotherapy-induced neuropathic pain during early life. Therefore, this study investigated the effects of VNC exposure between postnatal days (P) 11 and 21 on mechanical and thermal pain sensitivity in the developing rat. Low doses of VNC (15 or 30 μg/kg) failed to alter nociceptive withdrawal reflexes at any age examined compared with vehicle-injected littermate controls. Meanwhile, high dose VNC (60 μg/kg) evoked mechanical hypersensitivity in both sexes beginning at P26 that persisted until adulthood and included both static and dynamic mechanical allodynia. Hind paw withdrawal latencies to noxious heat and cold were unaffected by high doses of VNC, suggesting a selective effect of neonatal VNC on mechanical pain sensitivity. Gross and fine motor function appeared normal after VNC treatment, although a small decrease in weight gain was observed. The VNC regimen also produced a significant decrease in intraepidermal nerve fiber density in the hind paw skin by P33. Overall, the present results demonstrate that high-dose administration of VNC during the early postnatal period selectively evokes a mechanical hypersensitivity that is slow to emerge during adolescence, providing further evidence that aberrant sensory input during early life can have prolonged consequences for pain processing.


Individual or group antenatal education for childbirth/parenthood.
Structured antenatal education programs for childbirth and/or parenthood are commonly recommended for pregnant women and their partners by health care professionals in many parts of the world. Such programs are usually offered to groups but may be offered to individuals. The objective of this review was to assess the effects of this education on knowledge acquisition, anxiety, sense of control, pain, support, breastfeeding, infant care abilities, and psychological and social adjustment. The Cochrane Pregnancy and Childbirth Group trials register, the Cochrane Controlled Trials Register, and other databases were searched. The date of the last search was December, 1999. Randomized controlled trials of any structured educational program provided during pregnancy by an educator to either parent, that included information related to pregnancy, birth, or parenthood were included. The educational interventions could have been provided on an individual or group basis. Educational interventions directed exclusively to either increasing breastfeeding success or reducing smoking were excluded, since reviews of these topics can be found elsewhere in The Cochrane Library. Trial quality was assessed and data were extracted by the author from published reports. Six trials, involving 1443 women, were included. Twenty-two were excluded. The largest of the included studies (n = 1275) examined an educational intervention to increase vaginal birth after cesarean section. This high quality study showed similar rates of vaginal birth after cesarean section in 'verbal' and 'document' groups, relative risk (RR) 1.1 (95% confidence interval (CI) 1.0-1.2). More general educational interventions were the focus of the other five studies (combined n = 168). The methodological quality of these trials is uncertain, since details of the randomization procedure, allocation concealment, and/or participant accrual/loss were not reported. No consistent results were found. Sample sizes were very small, ranging from 10-67. Interventions, populations, and outcomes measured were different in each study. No data from the five general education trials were reported concerning labour and birth outcomes, anxiety, breastfeeding success, or general social support. Knowledge acquisition and factors related to infant care competencies were measured. Individualized prenatal education directed toward avoidance of a cesarean birth does not increase the rate of vaginal birth after cesarean section. The effects of general antenatal education for childbirth and/or parenthood remain unknown.


Underusers of mammogram screening: stage of adoption in five U.S. subpopulations. The NCI Breast Cancer Screening Consortium.
The purpose of this report is to describe the characteristics of women ages 50 to 80 who do not follow commonly accepted mammography screening guidelines. It provides unique understanding of the robustness of characteristics of underusers across five different U.S. subpopulations. The data are from the baseline surveys of the five studies of the NCI Breast Cancer Screening Consortium. Stage of adoption of mammography screening and other characteristics of underusers are presented. Polytomous logistic regression analysis was used to explore multivariable associations with stage of adoption in each study site. The five samples studied by the Consortium range in size from 259 to 4,477 women (n = 11,292). The relationship of the perceptions of the pros and cons of mammography with stage of adoption was strikingly similar across the five samples. Other variables consistently associated with stage were a recent receipt of a breast physical examination and recommendation for mammography by a physician. The findings suggest a need to encourage regular screening through effective communication from a health care provider. Intervention messages should be designed to increase the pros of mammography, decrease the cons, and highlight these differentially according to the woman's stage of adoption.


The New York State universal newborn hearing screening demonstration project: ages of hearing loss identification, hearing aid fitting, and enrollment in early intervention.
To determine the ages of hearing loss identification, hearing aid fitting, and enrollment in early intervention through a multi-center, state-wide universal newborn hearing screening project. Universal newborn hearing screening was conducted at eight hospitals across New York State. All infants who did not bilaterally pass hearing screening before discharge were recalled for outpatient retesting. Inpatient screening and outpatient rescreening were done with transient evoked otoacoustic emissions and/or auditory brain stem response testing. Diagnostic testing was performed with age appropriate tests, auditory brain stem response and/or visual reinforcement audiometry. Infants diagnosed with permanent hearing loss were considered for hearing aids and early intervention. Ages of hearing loss identification, hearing aid fitting, and enrollment in early intervention were investigated regarding nursery type, risk status, unilateral versus bilateral hearing loss, loss type, loss severity, and state regions. The prevalence of infants diagnosed with permanent hearing loss was 2.0/1000 (85 of 43,311). Of the 85 infants with hearing loss, 61% were from neonatal intensive care units (NICUs) and 67% were at risk for hearing loss. Of the 36 infants fitted with hearing aids, 58% were from NICUs and 78% were at risk for hearing loss. The median age at identification and enrollment in early intervention was 3 mo. Median age at hearing aid fitting was 7.5 mo. Median ages at identification were less for infants from the well-baby nurseries (WBNs) than for the NICU infants and for infants with severe/profound than for infants with mild/moderate hearing loss, but were similar for not-at-risk and at-risk infants. Median ages at hearing aid fitting were less for well babies than for NICU infants, for not-at-risk infants than for at-risk infants, and for infants with severe/ profound hearing loss than for infants with mild/ moderate hearing loss. However, median ages at early intervention enrollment were similar for nursery types, risk status, and severity of hearing loss. Early ages of hearing loss identification, hearing aid fitting, and enrollment in early intervention can be achieved for infants from NICUs and WBNs and for infants at risk and not at risk for hearing loss in a large multi-center universal newborn hearing screening program.


The value of unenhanced scans in differentiating lesions producing ring enhancement.
Ring enhancement with contrast material is a nonspecific computed tomographic finding seen in a variety of lesions, including benign and malignant neoplasms, inflammation, infarction, and hematoma. This lack of specificity is especially troublesome in the differentiation of self-limiting disorders (infarction and hematoma) from progressive processes (tumor or abscess) potentially requiring surgical treatment. To determine whether more specific diagnoses were possible, 115 cases with ring lesions were retrospectively evaluated for the usefulness of precontrast scan features. The presence or absence of a complete ring proved useful. A complete ring on unenhanced scans effectively excluded self-limiting processes; it was seen in none of 18 infarcts or 11 hematomas. A complete ring on unenhanced scans occurred in 37 of 65 neoplasms, and was more common in metastatic disease (2:1) than in gliomas. A complete ring on unenhanced computed tomography was also seen in four of 15 pyogenic abscesses.


Clinicopathologic aspects of 8 Kaposi's sarcomas among 1009 renal transplant patients.
In 8 of 1009 patients receiving renal grafts and consecutive immunosuppressive therapy during the last 20 years in Budapest, Hungary, Kaposi's sarcoma developed 6.5 months (average) after transplantation. Of 8 cutaneous cases of this disease, 5 also presented visceral manifestations. Seven patients died. Those with visceral tumors died because of the tumor itself. One patient affected by cutaneous tumor has survived for 5 years post-treatment. The relatively high incidence of Kaposi's sarcoma may be explained by a high infection rate with Herpes virus 8, which was shown to be positive in all three cases investigated.


Connecting the Dots between Barriers to W.I.C. Access and Adult and Child Food Insecurity: A Survey of Missouri Residents.
Background. Previous research has explored the impact of W.I.C. on recipients' health, but less is known about the connection between barriers to W.I.C. access and health outcomes. We fill in a gap in the literature by studying the relationship between barriers to Special Supplemental Nutrition Program for Women, Infants, and Children (W.I.C.) access and adult and child food insecurity. Methods. After survey administration, we analyzed a cross-sectional sample of 2244 residents in Missouri who have used W.I.C. or lived in a household with a W.I.C. recipient in the past three years. We ran logistic regression models to understand the relationships among barriers to W.I.C. utilization, adult food insecurity, and child food insecurity. Results. Having special dietary needs (for adults), lacking access to technology, encountering inconvenient clinic hours of operation, and experiencing difficulties taking off work were associated with increased adult food insecurity. Difficulties finding WIC-approved items in the store, technological barriers, inconvenient clinic hours, difficulties taking off work, and finding childcare were associated with increased child food insecurity. Conclusion. Barriers to accessing and utilizing W.I.C. are associated with adult and child food insecurity. However, current policies suggest promising approaches to curbing these barriers.


Improved Serodiagnostic Performance for Lyme Disease by Use of Two Recombinant Proteins in Enzyme-Linked Immunosorbent Assay Compared to Standardized Two-Tier Testing.
The most reliable test method for the serological confirmation of Lyme disease (LD) is a 2-tier method recommended by the CDC in 1995. The first-tier test is a low-specificity enzyme-linked immunosorbent assay (ELISA), and the second-tier tests are higher-specificity IgG and IgM Western blots. This study describes the selection of two Borrelia burgdorferi recombinant proteins and evaluation of their performance in a simple 1-tier test for the serological confirmation of LD. These two proteins were generated from (i) the full-length dbpA gene combined with the invariable region 6 of the vlsE gene (DbpA/C6) and (b) the full-length ospC gene (OspC). The expressed DbpA/C6 and OspC proteins were useful in detecting anti-Borrelia IgG and IgM antibodies, respectively. A blind study was conducted on a well-characterized panel of 279 human sera from the CDC, comparing ELISAs using these two recombinant antigens with the 2-tier test method. The two methods (DbpA/C6-OspC versus 2-tier test) were equivalent in identifying sera from negative-control subjects (99% and 100% specificity, respectively) and in detecting stage II and III LD patient sera (100% and 100% sensitivity). However, the DbpA/C6-OspC ELISA was markedly better (80% versus 63%) than the 2-tier test method in detecting anti-Borrelia antibodies in stage I LD patients. The findings suggest that these antigens could be used in a simple 1-tier ELISA that is faster to perform, easier to interpret, and less expensive than the 2-tier test method and which is better at detecting Borrelia-specific antibodies in sera from patients with stage I LD.


New delivery systems for local anaesthetics-part 2.
Part 2 of this paper deals with the techniques for drug delivery of topical and injectable local anaesthetics. The various routes of local anaesthetic delivery (epidural, peripheral, wound catheters, intra-nasal, intra-vesical, intra-articular, intra-osseous) are explored. To enhance transdermal local anaesthetic permeation, additional methods to the use of an eutectic mixture of local anaesthetics and the use of controlled heat can be used. These methods include iontophoresis, electroporation, sonophoresis, and magnetophoresis. The potential clinical uses of topical local anaesthetics are elucidated. Iontophoresis, the active transportation of a drug into the skin using a constant low-voltage direct current is discussed. It is desirable to prolong local anaesthetic blockade by extending its sensory component only. The optimal release and safety of the encapsulated local anaesthetic agents still need to be determined. The use of different delivery systems should provide the clinician with both an extended range and choice in the degree of prolongation of action of each agent.


Muscle fiber phenotype: a culprit of abnormal metabolism and function in skeletal muscle of humans with obesity.
The proportion of the different types of fibers in a given skeletal muscle contributes to its overall metabolic and functional characteristics. Greater proportion of type I muscle fibers is associated with favorable oxidative metabolism and function of the muscle. Humans with obesity have a lower proportion of type I muscle fibers. We discuss how lower proportion of type I fibers in skeletal muscle of humans with obesity may explain metabolic and functional abnormalities reported in these individuals. These include lower muscle glucose disposal rate, mitochondrial content, protein synthesis, and quality/contractile function, as well as increased risk for heart disease, lower levels of physical activity, and propensity for weight gain/resistance to weight loss. We delineate future research directions and the need to examine hybrid muscle fiber populations, which are indicative of a transitory state of fiber phenotype within skeletal muscle. We also describe methodologies for precisely characterizing muscle fibers and gene expression at the single muscle fiber level to enhance our understanding of the regulation of muscle fiber phenotype in obesity. By contextualizing research in the field of muscle fiber type in obesity, we lay a foundation for future advancements and pave the way for translation of this knowledge to address impaired metabolism and function in obesity.


Cubic liquid-crystalline nanoparticles.
Cubic liquid-crystalline nanoparticles prepared from aqueous dispersions of cubic lipid-water phases are kinetically stable in the presence of certain dispersing agents. The properties of cubic nanoparticles from monoolein-water and other lipid-water systems have been examined with a variety of experimental techniques. The cubic nanoparticles can be stabilized by polymerization of the reactive lipids in cubic lipid assemblies. Several low-energy-input methods have been developed to facilitate the production and application of cubic nanoparticles. The ability to incorporate and deliver lipophilic, amphiphilic, and water-soluble molecules in a controlled manner and the good biocompatibility of cubic nanoparticles make them excellent candidates for drug-delivery applications.


Intracellular cholesterol trafficking is dependent upon NPC2 interaction with lysobisphosphatidic acid.
Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein.


The Hemochron Response RxDx heparin and protamine dosing system.
The use of dosing assays to calculate heparin and protamine dose requirements during cardiac surgery has been shown to significantly improve overall postoperative patient outcome. When patients are managed with an individualized dosing system, intraoperative and postoperative transfusion requirements and bleeding are reduced. The Hemochron RxDx system is widely used as a complement to traditional activated clotting time testing to optimize anticoagulation management. The system consists of the heparin response test, the protamine response test, and the protamine dose assay. All are modifications of the activated clotting time using either Celite (Celite Corporation, Santa Barbara, CA) or kaolin as the activator. Dosing is calculated manually using earlier version Hemochron instruments (model 801) or automatically with the Hemochron 8000 or with the early versions of the Hemochron Response and the personal digital assistant (PDA) RxDx calculator. Missing from available user options is an automated RxDx system for the Response. A study was conducted at four clinical sites to compare recently developed Response RxDx software, which eliminates the need for the PDA RxDx calculator, to the existing Hemochron 8000 RxDx and to the Response-PDA RxDx systems. Similar to the current system, the operator inputs the patient's height, weight, and gender, and the software automatically calculates the blood volume. Using the clotting times determined on the Response, bolus heparin and protamine doses and any additional heparin and protamine requirements are calculated automatically. Data were collected from 76 patients, of which, 64 patients were on pump, 11 patients were off pump, and 1 patient was converted from off to on pump. The Response estimated blood volume calculations showed a correlation coefficient of 0.989 when compared with available systems. A good correlation was also observed for the bolus heparin (r = 0.925) and protamine doses (r = 0.900) with equivalence confirmed by a paired student's t test. These data confirm that the Response RxDx system yields results that are identical (P > 0.05) to those obtained using the Hemochron 8000 RxDx or Response-PDA RxDx calculator. The Response RxDx also offers expanded user options related to blood volume limits, expanded clotting time ranges for presetting default values, and flexibility in test sequence. Case records can be printed or downloaded to a PC via the HRDM data management program. The Hemochron Response RxDx represents a complete anticoagulation management system for the cardiac surgical patient.


Overview of acute type A dissection in Japan.
Acute type A aortic dissection is a relatively uncommon but devastating disease and usually requires emergency surgery. Based on the several database projects, a large amount of perioperative patient data has now been accumulated and is expected to be useful in clinical practice. Especially in Japan, the number of surgeries for acute type A aortic dissection has been gradually increasing recently, and the overall mortality rate has stabilized at less than 10%. One of the keys to further improvement in outcomes will be to improve the results of aortic root replacement. In addition, strategies need to be established for very elderly patients, comatose patients, and patients with malperfusion as preoperative conditions. The use of a relatively new device, the frozen elephant trunk, is also increasing and might be changing the surgical outcome. In this report, we describe the current status of acute type A aortic dissection in Japan, with reference to recent guidelines and literature.


Biologicals targeting T helper cell subset differentiating cytokines are effective in the treatment of murine anti-myeloperoxidase glomerulonephritis.
Anti-myeloperoxidase nephritogenic autoimmunity induces severe glomerulonephritis. To assess the therapeutic potential of monoclonal antibodies targeting T helper (Th) subset differentiation determining cytokines, we studied a murine model of anti-myeloperoxidase glomerulonephritis. The temporal participation of T helper subsets was determined by quantitating gene expression of CD4+ T-cells isolated from nephritic kidneys and cytokine production by lymphocytes from nodes draining myeloperoxidase immunization sites. Th17 cytokines (IL-17A and IL-6) rose rapidly but declined as autoimmunity matured when Th1 cytokines (IL-12 and TNF) predominated. Therefore, T helper subset participation in anti-myeloperoxidase autoimmunity is biphasic, with Th17 early and Th1 late. To confirm the functional relevance of this biphasic pattern, we compared systemic anti-myeloperoxidase autoimmunity in wild type, Th17 deficient and Th1 deficient mice. Early, Th1 deficient mice developed similar autoimmunity and glomerulonephritis to wild type mice. However, Th17 deficient mice had significantly reduced anti-myeloperoxidase autoimmunity. In late autoimmunity, Th1 deficient mice developed reduced autoimmunity and were protected from anti-myeloperoxidase glomerulonephritis. The therapeutic potential of these findings were demonstrated by neutralizing monoclonal antibodies. Targeting IL-23p19 attenuated early Th17 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not late phase disease. Targeting IL-12p35 attenuated late phase Th1 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not early autoimmunity or glomerulonephritis. Targeting both T helper subsets with an anti-IL-12p40 monoclonal antibody was effective during both early and late phases of anti-myeloperoxidase glomerulonephritis. Thus, definition of dominant T helper differentiating subsets in anti-myeloperoxidase glomerulonephritis by renal CD4+ T-cell cytokine gene expression allows effective proper phase monoclonal antibody treatment of anti-myeloperoxidase glomerulonephritis.


Positive impact of increased number of emergency consultants.
The increased presence of consultant staff should theoretically lead to better outcomes in emergency departments (EDs). A retrospective observational study was conducted in a tertiary paediatric emergency department (PED) over a 10-year period documenting trends in percentage of children admitted, complaints to the department and average waiting times. Consultant numbers increased from 2.6 to 6.2 full time equivalent staff between 2000 and 2004. Other staffing numbers were essentially unchanged. All parameters examined improved coincident with increasing consultant numbers. The percentage of children admitted decreased by 27%, complaints fell by 41% and the average waiting time by 15%. The yearly cost of an additional 3.6 consultants (2005) was $A1,003,490 with net saving to the hospital of over $A9.48 million. The provision of additional consultant medical staff in a PED coincided with a decrease in the percentage of children admitted, complaints to the department and average waiting times, and was cost effective.


Reduced inflammation accompanies diminished myelin damage and repair in the NG2 null mouse spinal cord.
Multiple sclerosis (MS) is a demyelinating disease in which blood-derived immune cells and activated microglia damage myelin in the central nervous system. While oligodendrocyte progenitor cells (OPCs) are essential for generating oligodendrocytes for myelin repair, other cell types also participate in the damage and repair processes. The NG2 proteoglycan is expressed by OPCs, pericytes, and macrophages/microglia. In this report we investigate the effects of NG2 on these cell types during spinal cord demyelination/remyelination. Demyelinated lesions were created by microinjecting 1% lysolecithin into the lumbar spinal cord. Following demyelination, NG2 expression patterns in wild type mice were studied via immunostaining. Immunolabeling was also used in wild type and NG2 null mice to compare the extent of myelin damage, the kinetics of myelin repair, and the respective responses of OPCs, pericytes, and macrophages/microglia. Cell proliferation was quantified by studies of BrdU incorporation, and cytokine expression levels were evaluated using qRT-PCR. The initial volume of spinal cord demyelination in wild type mice is twice as large as in NG2 null mice. However, over the ensuing 5 weeks there is a 6-fold improvement in myelination in wild type mice, versus only a 2-fold improvement in NG2 null mice. NG2 ablation also results in reduced numbers of each of the three affected cell types. BrdU incorporation studies reveal that reduced cell proliferation is an important factor underlying NG2-dependent decreases in each of the three key cell populations. In addition, NG2 ablation reduces macrophage/microglial cell migration and shifts cytokine expression from a pro-inflammatory to anti-inflammatory phenotype. Loss of NG2 expression leads to decreased proliferation of OPCs, pericytes, and macrophages/microglia, reducing the abundance of all three cell types in demyelinated spinal cord lesions. As a result of these NG2-dependent changes, the course of demyelination and remyelination in NG2 null mice differs from that seen in wild type mice, with both myelin damage and repair being reduced in the NG2 null mouse. These studies identify NG2 as an important factor in regulating myelin processing, suggesting that therapeutic targeting of the proteoglycan might offer a means of manipulating cell behavior in demyelinating diseases.


[Pharmacological correction of alterations of apoptosis of the neurons of the hypothalamus suprachiasmatic nucleus and pinealocytes during aging and stress.]
As is known, the pineal gland plays an important role in adaptogenesis, and the hypothalamus is one of the main links of the stress-reactive system and is involved in the regulation of the involution of the whole organism. So, the study of changes in these organs during stress and aging is very interesting. The aim of the work is to study the mechanisms of apoptosis of pinealocytes and neurosecretory cells of the suprachiasmatic nucleus of the hypothalamus during aging, stress, and under the conditions of pharmacological correction of involutional processes and stress response (antioxidant alpha-tocopherol acetate, immunomodulator cycloferon). We used Wistar rats as model, young (2-4 months) and old (30 months). Age-related features of the apoptosis dynamics of pinealocytes and neurosecretory cells of the hypothalamic suprachiasmatic nucleus were studied using TUNEL and immunohistochemistry, and the possibilities of pharmacological correction of apoptotic processes are determined. An age-dependent increase of apoptosis level of cells of suprachiasmatic nucleus and epiphysis in rats was revealed. The stress effect (immobilization) led to the intensification of cell death, more significant in older animals. The pineal gland and suprachiasmatic nucleus, traditionally regarded as regulators of circadian rhythms, are at the same time actively involved in general adaptation processes. The studied drugs (α-tocopherol-acetate, cycloferon, and their combination) have a pronounced anti-apoptotic, cytoprotective effect under physiological conditions during aging, as well as during non-specific emotional stress (immobilization) in young and old animals. The regulatory effect is accomplished by activating the expression of the anti-apoptotic protein Bcl-2 in the neurosecretory cells of the suprachiasmatic nucleus and pinealocytes.


The spatial and temporal organization of origin firing during the S-phase of fission yeast.
Eukaryotes duplicate their genomes using multiple replication origins, but the organization of origin firing along chromosomes and during S-phase is not well understood. Using fission yeast, we report the first genome-wide analysis of the spatial and temporal organization of replication origin firing, analyzed using single DNA molecules that can approach the full length of chromosomes. At S-phase onset, origins fire randomly and sparsely throughout the chromosomes. Later in S-phase, clusters of fired origins appear embedded in the sparser regions, which form the basis of nuclear replication foci. The formation of clusters requires proper histone methylation and acetylation, and their locations are not inherited between cell cycles. The rate of origin firing increases gradually, peaking just before mid S-phase. Toward the end of S-phase, nearly all the available origins within the unreplicated regions are fired, contributing to the timely completion of genome replication. We propose that the majority of origins do not fire as a part of a deterministic program. Instead, origin firing, both individually and as clusters, should be viewed as being mostly stochastic.


Nematocera flies recorded in Serra do Courel, northwest Spain, May 2012 (Diptera: Anisopodidae, Blepharoceridae, Cylindrotomidae, Limoniidae, Pediciidae, Tipulidae and Trichoceridae) including descriptions of two new species of Limoniidae.
During May 2012 Diptera were sampled in the Serro do Courel area of Lugo Province, Galicia, northwest Spain. The authors of this paper, members of the Malloch Society (see website) are active in attempting to understand the detailed ecology of flies. Much of this work is through targeting larval stages often with an emphasis on saproxylic situations. By rearing adults from larvae direct relationships between them and their detailed habitat requirements are established. The list of nematocerous Diptera that were sampled includes 36 species two of them new to science and records of six others new to the Iberian peninsula are provided. We describe Lipsothrix galiciensis Hancock & Hewitt sp. nov., and Prionolabis pjotri Hancock sp. nov. of the family Limoniidae and provide a key to adults of European Lipsothrix species. Such results from this brief opportunity indicate the potential of the area for further field work in these and other families of Diptera.


Anticholinesterase activity of phenolic acids and their derivatives.
The ability of 36 phenolic acids and their derivatives to inhibit acetyl- and butyrylcholinesterase was studied. The most efficient acetylcholine inhibitors were: carnosic acid = gentisic acid > 3-hydroxy-4-methoxycinnamic acid = ethyl ferulate = ethyl vanillate = nordihydroguaiaretic acid > ethyl 4-hydroxybenzoate = methyl ferulate. The order of effectiveness towards butyrylcholinesterase was: carnosic acid > nordihydroguaiaretic acid = ethyl ferulate > salicylic acid > gentisic acid > rosmarinic acid = caftaric acid > homogentisic acid. The inhibitory activity was dependent on the number/position of OH or/and OCH3 groups attached to a phenol ring. It can be speculated that OCH3 substitution in the phenol ring can promote a higher antibutyrylcholinesterase activity (although not statistically confirmed at p < 0.05). The presence of a CH=CH-COOH group had a highly favourable effect on the antiacetylcholinesterase activity compared with a CH2-CH2-COOH or a COOH group. Methyl and ethyl esters were more potent inhibitors than the corresponding free acids. The molecular weight of the compounds (in the range of M = 154.12 - 474 g/mol) played a minor role in this context.


Molecular Changes In Cardiac Tissue As A New Marker To Predict Cardiac Dysfunction Induced By Radiotherapy.
The contribution of radiotherapy, per se, to late cardiotoxicity remains controversial. To clarify its impact on the development of early cardiac dysfunction, we developed an experimental model in which the hearts of rats were exposed, in a fractionated plan, to clinically relevant doses of ionizing radiation for oncological patients that undergo thoracic radiotherapy. Rat hearts were exposed to daily doses of 0.04, 0.3, and 1.2 Gy for 23 days, achieving cumulative doses of 0.92, 6.9, and 27.6 Gy, respectively. We demonstrate that myocardial deformation, assessed by global longitudinal strain, was impaired (a relative percentage reduction of >15% from baseline) in a dose-dependent manner at 18 months. Moreover, by scanning electron microscopy, the microvascular density in the cardiac apex was significantly decreased exclusively at 27.6 Gy dosage. Before GLS impairment detection, several tools (qRT-PCR, mass spectrometry, and western blot) were used to assess molecular changes in the cardiac tissue. The number/expression of several genes, proteins, and KEGG pathways, related to inflammation, fibrosis, and cardiac muscle contraction, were differently expressed in the cardiac tissue according to the cumulative dose. Subclinical cardiac dysfunction occurs in a dose-dependent manner as detected by molecular changes in cardiac tissue, a predictor of the severity of global longitudinal strain impairment. Moreover, there was no dose threshold below which no myocardial deformation impairment was detected. Our findings i) contribute to developing new markers and exploring non-invasive magnetic resonance imaging to assess cardiac tissue changes as an early predictor of cardiac dysfunction; ii) should raise red flags, since there is no dose threshold below which no myocardial deformation impairment was detected and should be considered in radiation-based imaging and -guided therapeutic cardiac procedures; and iii) highlights the need for personalized clinical approaches.


Helping pregnant women make better decisions: a systematic review of the benefits of patient decision aids in obstetrics.
Objectives Patient decision aids can be used to support pregnant women engaging in shared decisions, but little is known about their effects in obstetrics. The authors aimed to evaluate the effects of patient decision aids designed for pregnant women on clinical and psychosocial outcomes. Design Systematic review. Data on all outcomes were extracted and summarised. All studies were critically appraised for potential sources of bias and, when possible to obtain, the reported decision aids were evaluated. Meta-analysis was not possible due to the heterogeneity of outcomes in primary studies and the small number of studies. Data sources Electronic searches were performed using Medline, Embase, the Cochrane Library and Medion databases from inception until December 2010. Reference lists of all included articles were also examined and key experts contacted. Eligibility criteria for selecting studies Eligibility criteria included randomised controlled trials, which reported on patient decision aids for women facing any treatment decision in pregnancy published in English. Studies evaluating health education material that did not address women's values and preferences were excluded. Results Patient decision aids have been developed for decisions about prenatal testing, vaginal birth after Caesarean section, external cephalic version and labour analgesia. Use of decision aids is associated with a number of positive effects including reduced anxiety, lower decisional conflict, improved knowledge, improved satisfaction and increased perception of having made an informed choice. Conclusions Patient decision aids have the potential to improve obstetric care. However, currently the evidence base is limited by the small number of studies, the quality of the studies and because they involved heterogeneous decision aids, patient groups and outcomes.


The Microcirculation Physiome Project.
Presented is a discussion of steps towards the creation of a database of the microcirculation encompassing anatomical and functional experimental data, and conceptual and computational models. The discussion includes issues of database utility, organization, data deposition, and linkage to other databases. The database will span levels from gene to tissue and will serve both research and educational purposes.


Financing clinical dental education.
Many reports have documented the growing financial challenges faced by dental schools. This article examines the financial implications of two new models of dental education: 1) seniors spend 70 percent of their time in community clinics and practices, providing general dental care to underserved patients, and 2) schools develop patient-centered clinics where teams of faculty, residents, and senior students provide care to patients. We estimate that the average dental school will generate new net revenues of about $2.7 million per year from the community-based educational programs for senior students and about $14 million per year from patient-centered care clinics. These are upper boundary estimates and vary greatly by school. The organizational and financial challenges of moving to these new educational models are discussed.


The combination of granulocyte-monocyte apheresis and vedolizumab: A new treatment option for ulcerative colitis?
To assess the effectiveness and safety of combining granulocyte-monocyte apheresis (GMA) and vedolizumab (VDZ) in patients with refractory ulcerative colitis (UC). This retrospective, multicentre pilot study included all UC patients receiving both GMA and VDZ. We recorded data on GMA sessions, demographic characteristics, and clinical response. Effectiveness was assessed 1 and 6 months after finishing the GMA using the partial Mayo score, C-reactive protein, and fecal calprotectin levels. Data were also compiled on VDZ intensification, use of new immunomodulators and colectomy during follow-up. Eight patients were included (mean age 46 years; 63% female; mean disease duration, 132 months; 50% E3). GMA was started after a loss of response to VDZ in all cases (25% primary nonresponse and 75% secondary loss of response). All had previously received anti-TNF agents. VDZ was prescribed as the second-, third-, or fourth-line biologic in 37%, 50%, and 13% of cases, respectively. Patients had a mean baseline partial Mayo score of 7.5 (SD 2.1) and received a median of 15 GMA sessions (range 5-38). After a median follow-up of 7.5 months (IQR 5-12), partial Mayo score decreased after 1 and 6 months (P = .01 and .06, respectively). Three patients (38%) achieved steroid-free clinical remission and five (63%) withdrew VDZ. Colectomy rate was 38%. No adverse events were observed during the combination therapy. This small case series suggests that combining GMA with VDZ could be a treatment option in selected cases of UC with an inadequate response to this biologic agent.


Heat stability of carboxypeptidase R of experimental animals.
Carboxypeptidase N (CPN) and carboxypeptidase R (CPR) are present in fresh serum, and cleave C-terminal arginine or lysine residues from bioactive peptides such as anaphylatoxins and kinins resulting in regulation of peptide activity. Although CPN is present in the active form in plasma, CPR is generated from proCPR by trypsin-like enzymes such as thrombin. CPR regulates not only inflammatory peptides but also restricts fibrinolysis. To elucidate the complex role of CPN and CPR in vivo, studies in animal models will be essential. CPR of guinea pig, rat and rabbit decayed at 37 C rapidly as in the case of human CPR. However, at 25 C, CPR of those species decayed to some extent, although human serum CPR did not decay within 60 min. In the presence of thrombin inhibitor, CPR in the sera of animals tested decayed more rapidly than CPR in serum without thrombin inhibitor suggesting that additional generation of CPR may have been prevented during decay evaluation. However, human serum CPR decayed more rapidly in the absence of thrombin inhibitor indicating that thrombin may accelerate the decay in human serum.


Distribution and significance of norepinephrine in the lateral cochlear wall of pigmented and albino rats.
Picogram quantities of norepinephrine were found in cochlear regions of pigmented and non-pigmented rats. These regions of the cochlea were the modiolus, organ of Corti--osseous spiral lamina and the lateral cochlear wall. The content of norepinephrine in the modiolus and lateral cochlear wall of the pigmented rat was significantly greater than that in areas of the non-pigmented rat. In contrast, there was no statistical difference between the norepinephrine content of the organ of Corti--osseous spiral lamina region of the pigmented rat and that of the albino rat. Since a major difference between the pigmented and albino rats is the presence of melanin-containing melanocytes in the modiolus and lateral cochlear wall region of the pigmented animals, it is possible that norepinephrine is stored in cochlear melanocytes.


A bispecific nanobody to provide full protection against lethal scorpion envenoming.
Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100-kDa horse plasma antivenom serum (PAS)-derived Fab'(2) to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single-domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI' toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI'-toxin neutralization was monitored in mice. Remarkably, the NbAahI'F12 fully protected mice against 100 LD(50) of AahI' administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD(50) of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI'/AahII toxins succeeded in neutralizing 5 LD(50). Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 microg of NbF12-10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12-10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.


The enigma of 7q36 linked autosomal dominant limb girdle muscular dystrophy.
Two families with autosomal dominant limb girdle muscular dystrophy (LGMD) have previously been linked to a locus on chromosome 7q36 10 years ago. The locus has been termed both LGMD1D and 1E, but because of lack of additional families to narrow down the linked region of interest, this disease has remained elusive. A large Finnish family was clinically and genetically investigated. Laboratory parameters were determined, including creatine kinase (CK) value, neurographic and electromyography studies, cardiac and respiratory function examinations, muscle biopsies and muscle imaging by CT or MRI. Patients had onset of muscle weakness in the pelvic girdle between the fourth and sixth decades with an autosomal dominant pattern of inheritance. CK values were slightly elevated and electromyography was myopathic only. Muscle biopsies showed myopathic and/or dystrophic features with very minor rimmed vacuolation and protein aggregation findings. Molecular genetic analysis indicates linkage of the disease to the locus on chromosome 7q36 completely overlapping with the previously reported locus LGMD1D/E. Advancement towards the causative gene defect in the 7q36 linked disease needs new additional families to narrow the region of interest. The phenotype in the previously linked families has not been reported in full detail, which may be one reason for the shortage of additional families. A comprehensive clinical and morphological phenotype of chromosome 7q36 linked autosomal dominant LGMD with a restricted and updated 6.4 Mb sized haplotype is reported here.


An autocrine axis in the testis that coordinates spermiation and blood-testis barrier restructuring during spermatogenesis.
The mechanism(s) that regulate and coordinate the events of spermiation and blood-testis barrier (BTB) restructuring in the seminiferous epithelium that occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis are unknown. In this report, fragments derived from the laminin complex composed of laminin alpha3, beta3, and gamma3 chains (laminin-333) at the apical ectoplasmic specialization (apical ES) were shown to modulate BTB dynamics directly and/or indirectly via hemidesmosome. Experiments were performed using cultured Sertoli cells with functional tight junction (TJ) barrier and the ultrastructural features of the BTB but not apical ES. Recombinant protein fragments of laminin beta3 and gamma3 chains were shown to reduce the protein levels of occludin and beta1-integrin dose dependently at the Sertoli-Sertoli and Sertoli-basement membrane interface, respectively, thereby destabilizing the BTB permeability function. These results were corroborated by transient overexpression of laminin fragments in Sertoli cells. To further assess the role of beta1-integrin in hemidesmosome, knockdown of beta1-integrin in Sertoli cells by RNAi was found to associate with occludin redistribution at the Sertoli-Sertoli cell interface, wherein occludin moved away from the cell surface and became associated with endosomes, thereby destabilizing the BTB. In short, an apical ES-BTB-hemidesmosome autocrine regulatory axis was identified in testes, coordinating the events of spermiation and BTB restructuring that occur at the opposite ends of the seminiferous epithelium during spermatogenesis.


Needle exchange programs: an economic evaluation of a local experience.
To determine whether providing a needle exchange program to prevent HIV transmission among injection drug users would cost less than the health care consequences of not having such a program. Incidence outcome model to estimate the number of cases of HIV infection that this program would prevent over 5 years, assuming that the HIV incidence rate would be 2% with the program and 4% without it, and that an estimated 275 injection drug users would use the service over this time. Hamilton, Ont. Estimated number of cases of HIV infection expected to be prevented with and without the program over 5 years; estimated lifetime health care costs of treating an AIDS patient. The indirect costs of AIDS to society (e.g., lost productivity and informal caregiving) were not included. Projected costs were adjusted (discounted) to reflect their present value. In a sensitivity analysis, 3 parameters were varied: the estimate of the HIV transmission rate if no needle exchange program were provided, the number of injection drug users participating in the program, and the discount rate. With very conservative estimates, it was predicted that the Hamilton needle exchange program will prevent 24 cases of HIV infection over 5 years, thereby providing cost savings of $1.3 million after the program costs are taken into account. This translates into a ratio of cost savings to costs of 4:1. The sensitivity analysis confirmed that these findings are robust. Needle exchange programs are an efficient use of financial resources.


[Cecal hernia through Winslow's foramen. Radiographic study of a case and review of the literature].
A case of herniation of the cecum through the foramen of Winslow is reported in a 49-year-old man, admitted for acute abdominal pain. Diagnosis, suspected by abdominal plain films, and established by water-soluble enema, was confirmed by emergency surgical management. Herniation through Winslow's foramen represents the least common variety of internal hernias. Only 136 observations were previously reported, the cecum being involved in 25-30% of cases. Radiological diagnosis was made in only one patient out of ten. Although rare in occurrence, it carries a high mortality risk when diagnosis and treatment are delayed. A better appreciation of the classic radiological features will allow for earlier recognition and treatment. Therefore, the authors emphasize the interest of radiologic findings in this condition.


Density functional investigation of the Mitsunobu reaction.
In this article we performed an extensive density functional [BP86/6-311++G(3df,3pd) level] investigation of the hypersurface of the Mitsunobu reaction. Reaction of a phosphine with a dialkyl azodicarboxylate (first step in the Mitsunobu conversion) leads to either a five-membered oxadiazaphosphole ring (more stable) or a betaine. The subsequent formation of two stable intermediates, a dialkoxyphosphorane and an acyloxyalkoxyphosphorane, constitutes the second step in the mechanism. These intermediates are in equilibrium with each other (under exchange of alkoxy and acyloxy ligands), and both can undergo an acid-induced decomposition to yield the alkoxy- and/or acyloxyphosphonium salts. The alkoxyphosphonium salt generates the desired ester via a SN2 mechanism (inversion product). Alternatively, the phosphorus atom in a mixed acyloxyalkoxyphosphorane species can easily undergo Berry pseudorotation. A subsequent intramolecular substitution leads to the final ester via a retention mechanism. The hypersurface is much more complicated than previously assumed, and the Mitsunobu reaction is fundamentally capable of running under either inversion or retention. The possibility of selective stereocontrol is discussed. Side reactions include the formation of a degradation product and an anhydride.


A germ line mutation in exon 5 of the p53 gene in an extended cancer family.
Germ line p53 point mutations have been reported for some families with Li-Fraumeni syndrome, a syndrome characterized by a dominantly inherited increased susceptibility for the development of early age of onset neoplasms of diverse origin in multiple family members. All of the initially reported p53 germ line mutations have been found exclusively within a single conserved, nonpolymorphic region of the gene between condons 245 and 258. The restricted distribution of these inherited mutations has led to speculation that germ line p53 mutations have unique properties [B. Vogelstein, Nature (Lond.), 348: 681-682, 1990]. We report here on the identification of a p53 germ line mutation at codon 133 (ATG----ACG) in nine members of an extended Li-Fraumeni syndrome family. This mutation leads to an amino acid substitution in the protein and is shown to completely cosegregate with Li-Fraumeni syndrome associated cancer in this family. Its location extends the region of the p53 gene where inherited mutations predisposing to cancer are observed and suggests that their distribution may be diverse.


Mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apo)A-I from murine RAW 264.7 macrophages.
Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function. Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [3H]cholesterol to apolipoprotein (apo) A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, Gapdh, and combined with studies of this molecule on cholesterol esterification, de novo lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett's or Bonferroni post t-tests, as appropriate. The positive control, resveratrol (24 h), significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; p<0.01) and oligomycin (55%; p<0.01), under conditions (10 μM, 3 h) which did not induce cellular toxicity or deplete total cellular ATP content. Levels of ATP binding cassette transporter A1 (ABCA1) protein were repressed by oligomycin under optimal efflux conditions, despite paradoxical increases in Abca1 mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (Abca1, Abcg4, Stard1) and cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf2), indicating profound dysregulation of cholesterol homeostasis. Acute loss of mitochondrial function, and in particular Δψm, reduces cholesterol efflux to apoA-I and dysregulates macrophage cholesterol homeostasis mechanisms. Bioavailable antioxidants, targeted to mitochondria and capable of sustaining effective mitochondrial function, may therefore prove effective in maintenance of arterial health.


Issues and Perspectives in Species Delimitation using Phenotypic Data: Atlantean Evolution in Darwin's Finches.
Progress in the development and use of methods for species delimitation employing phenotypic data lags behind conceptual and practical advances in molecular genetic approaches. The basic evolutionary model underlying the use of phenotypic data to delimit species assumes random mating and quantitative polygenic traits, so that phenotypic distributions within a species should be approximately normal for individuals of the same sex and age. Accordingly, two or more distinct normal distributions of phenotypic traits suggest the existence of multiple species. In light of this model, we show that analytical approaches employed in taxonomic studies using phenotypic data are often compromised by three issues: 1) reliance on graphical analyses that convey little information on phenotype frequencies; 2) exclusion of characters potentially important for species delimitation following reduction of data dimensionality; and 3) use of measures of central tendency to evaluate phenotypic distinctiveness. We outline approaches to overcome these issues based on statistical developments related to normal mixture models (NMMs) and illustrate them empirically with a reanalysis of morphological data recently used to claim that there are no morphologically distinct species of Darwin's ground-finches (Geospiza). We found negligible support for this claim relative to taxonomic hypotheses recognizing multiple species. Although species limits among ground-finches merit further assessments using additional sources of information, our results bear implications for other areas of inquiry including speciation research: because ground-finches have likely speciated and are not trapped in a process of "Sisyphean" evolution as recently argued, they remain useful models to understand the evolutionary forces involved in speciation. Our work underscores the importance of statistical approaches grounded on appropriate evolutionary models for species delimitation. We discuss how NMMs offer new perspectives in the kind of inferences available to systematists, with significant repercussions on ideas about the phenotypic structure of biodiversity.


Transcriptome analysis reveals involvement of thiopurine S-methyltransferase in oxidation-reduction processes.
Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in the deactivation of thiopurines and represents a major determinant of thiopurine-related toxicities. Despite its well-known importance in thiopurine metabolism, the understanding of its endogenous role is lacking. In the present study, we aimed to gain insight into the molecular processes involving TPMT by applying a data fusion approach to analyze whole-genome expression data. The RNA profiling was done on whole blood samples from 1017 adult male and female donors to the Estonian biobank using Illumina HTv3 arrays. Our results suggest that TPMT is closely related to genes involved in oxidoreductive processes. The in vitro experiments on different cell models confirmed that TPMT influences redox capacity of the cell by altering S-adenosylmethionine (SAM) consumption and consequently glutathione (GSH) synthesis. Furthermore, by comparing gene networks of subgroups of individuals, we identified genes, which could have a role in regulating TPMT activity. The biological relevance of identified genes and pathways will have to be further evaluated in molecular studies.


The ratio of Treg/Th17 cells correlates with the disease activity of primary immune thrombocytopenia.
Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4(+) T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Treg cells to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio. Thirty adult patients with newly diagnosed ITP enrolled in this study. Twelve patients had been clinically followed up for 12 months. The percentages of CD4(+)CD25(hi)Foxp3(+) Treg cells and CD3(+)CD4(+)IL-17-producing Th17 cells in these patients and healthy controls (n = 17) were longitudinally analyzed by flow cytometry. The percentage of Treg cells in ITP patients was significantly lower than that of healthy controls, and the percentage of Th17 cells increased significantly at disease onset. The ratio of Treg/Th17 correlated with the disease activity. The ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients.


Intercepting virtual balls approaching under different gravity conditions: evidence for spatial prediction.
To accurately time motor responses when intercepting falling balls we rely on an internal model of gravity. However, whether and how such a model is also used to estimate the spatial location of interception is still an open question. Here we addressed this issue by asking 25 participants to intercept balls projected from a fixed location 6 m in front of them and approaching along trajectories with different arrival locations, flight durations, and gravity accelerations (0g and 1g). The trajectories were displayed in an immersive virtual reality system with a wide field of view. Participants intercepted approaching balls with a racket, and they were free to choose the time and place of interception. We found that participants often achieved a better performance with 1g than 0g balls. Moreover, the interception points were distributed along the direction of a 1g path for both 1g and 0g balls. In the latter case, interceptions tended to cluster on the upper half of the racket, indicating that participants aimed at a lower position than the actual 0g path. These results suggest that an internal model of gravity was probably used in predicting the interception locations. However, we found that the difference in performance between 1g and 0g balls was modulated by flight duration, the difference being larger for faster balls. In addition, the number of peaks in the hand speed profiles increased with flight duration, suggesting that visual information was used to adjust the motor response, correcting the prediction to some extent.NEW & NOTEWORTHY Here we show that an internal model of gravity plays a key role in predicting where to intercept a fast-moving target. Participants also assumed an accelerated motion when intercepting balls approaching in a virtual environment at constant velocity. We also show that the role of visual information in guiding interceptive movement increases when more time is available.


Quantitative genetics, version 3.0: where have we gone since 1987 and where are we headed?
The last 20 years since the previous World Congress have seen tremendous advancements in quantitative genetics, in large part due to the advancements in genomics, computation, and statistics. One central theme of this last 20 years has been the exploitation of the vast harvest of molecular markers--examples include QTL and association mapping, marker-assisted selection and introgression, scans for loci under selection, and methods to infer degree of coancestry, population membership, and past demographic history. One consequence of this harvest is that phenotyping, rather than genotyping, is now the bottleneck in molecular quantitative genetics studies. Equally important have been advances in statistics, many developed to effectively use this treasure trove of markers. Computational improvements in statistics, and in particular Markov Chain Monte Carlo (MCMC) methods, have facilitated many of these methods, as have significantly improved computational abilities for mixed models. Indeed, one could argue that mixed models have had at least as great an impact in quantitative genetics as have molecular markers. A final important theme over the past 20 years has been the fusion of population and quantitative genetics, in particular the importance of coalescence theory with its applications for association mapping, scans for loci under selection, and estimation of the demography history of a population. What are the future directions of the field? While obviously important surprises await us, the general trend seems to be moving into higher and higher dimensional traits and, in general, dimensional considerations. We have methods to deal with infinite-dimensional traits indexed by a single variable (such as a trait varying over time), but the future will require us to treat much more complex objects, such as infinite-dimensional traits indexed over several variables and with graphs and dynamical networks. A second important direction is the interfacing of quantitative genetics with physiological and developmental models as a step towards both the gene-phenotype map as well as predicting the effects of environmental changes. The high-dimensional objects we will need to consider almost certainly have most of their variation residing on a lower (likely much lower) dimensional subspace, and how to treat these constraints will be an important area of future research. Conversely, the univariate traits we currently deal with are themselves projections of more complex structures onto a lower dimensional space, and simply treating these as univariate traits can result in serious errors in understanding their selection and biology. As a field, our future is quite bright. We have new tools and techniques, and (most importantly) new talent with an exciting international group of vibrant young investigators who have received their degrees since the last Congress. One cloud for concern, however, has been the replacement at many universities of plant and animal breeders with plant and animal molecular biologists. Molecular tools are now an integral part of breeding, but breeding is not an integral part of molecular biology.


At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.
We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.


Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis).
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface.


Chitosan interaction with iron from yoghurt using an in vitro digestive model: comparative study with plant dietary fibers.
The objective of this work was to investigate the interaction of chitosan with iron from yoghurt by an in vitro gastrointestinal tract model. Taking into account that chitosan is a polysaccharide included in fiber definition by Codex Alimentarius; chitosan behavior was studied and compared with different plant fiber (wheat, bamboo, apple, psyllium and inulin) behaviors, in the same in vitro conditions. Ferrous sulfate was added to yoghurts with each type of fiber. The gastric environment was simulated with HCl (pH 1.0-2.0). The duodenal environment was simulated with NaHCO(3) (pH 6.8-7.2) and a dialysis tubing cellulose membrane. Results showed that chitosan had the highest iron retention percentages (53.2% at 30 min; 56.8% at 60 min) interacting in a more pronounced manner with iron than the plant fibers used in this work.


Does neighbourhood social capital aid in levelling the social gradient in the health and well-being of children and adolescents? A literature review.
Although most countries in the European Union are richer and healthier than ever, health inequalities remain an important public health challenge. Health-related problems and premature death have disproportionately been reported in disadvantaged neighbourhoods. Neighbourhood social capital is believed to influence the association between neighbourhood deprivation and health in children and adolescents, making it a potentially interesting concept for policymakers. This study aims to review the role of social capital in health inequalities and the social gradient in health and well-being of children and adolescents. A systematic review of published quantitative literature was conducted, focussing on (1) the mediating role of neighbourhood social capital in the relationship between socio-economic status (SES) and health-related outcomes in children and adolescents and (2) the interaction between neighbourhood social capital and socio-economic characteristics in relation to health-related outcomes in children and adolescents. Three electronic databases were searched. Studies executed between 1 January 1990 and 1 September 2011 in Western countries (USA, New Zealand, Australia and Europe) that included a health-related outcome in children or adolescents and a variable that measured neighbourhood social capital were included. Eight studies met the inclusion criteria for the review. The findings are mixed. Only two of five studies confirmed that neighbourhood social capital mediates the association between neighbourhood deprivation and health and well-being in adolescents. Furthermore, two studies found a significant interaction between neighbourhood socio-economic factors and neighbourhood social capital, which indicates that neighbourhood social capital is especially beneficial for children who reside in deprived neighbourhoods. However, two other studies did not find a significant interaction between SES and neighbourhood social capital. Due to the broad range of studied health-related outcomes, the different operationalisations of neighbourhood social capital and the conceptual overlap between measures of SES and social capital in some studies, the factors that explain these differences in findings remain unclear. Although the findings of this study should be interpreted with caution, the results suggest that neighbourhood social capital might play a role in the health gradient among children and adolescents. However, only two of the included studies were conducted in Europe. Furthermore, some studies focussed on specific populations and minority groups. To formulate relevant European policy recommendations, further European-focussed research on this issue is needed.


Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens.
The chemical composition of the outer membrane fractions (OMFs) of Eikenella corrodens strains 23834 and 470 as well as the strain 23834 lipopolysaccharide (LPS) was determined. The OMFs were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMFs of strains 23834 and 470, free of cytoplasmic membrane components, were found to contain 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate, and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis both OMFs contained one major peptide determined to be 33,500 daltons for the strain 23834 OMF, and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic, and lesser amounts of octadecanoic acids. Transmission electron microscopic examination of the OMFs revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was found to be composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose could be detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed the E. corrodens LPS to resemble other smooth-type LPSs. Transmission electron microscopic examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a "classical," i.e., enteric, type of LPS.


Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC-LptA Interaction and Decreasing LptA Stability.
The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC-LptA and LptA-LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC-LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.


[Viral infection, atherosclerosis, and ischemic heart disease].
Data are submitted on significant herpes viruses- and cytomegalovirus-associated infections in patients with unstable angina, which go beyond those in essentially healthy subjects of the same age. Taking into account strong impact the viral infection exerts on immune reactions it will be noted that virus-infected subjects are much sensitive to the action of autoantigens and furthermore, that autoimmune inflammation plays its part in the development of unstable angina.