Choline, phosphatidylcholine and sphingomyelin in human and bovine milk and infant formulas.
Choline is a precursor for the biosynthesis of phosphatidylcholine (lecithin), sphingomyelin, and choline plasmalogens--all essential constituents of membranes. Choline is also needed to make acetylcholine, a major neurotransmitter. The major choline-containing compounds of human milk (unesterified choline, phosphatidylcholine, sphingomyelin) were measured in samples obtained from mothers of full-term infants. Unesterified choline concentrations were highest (greater than 600 nmol/ml) during the first week of lactation, but thereafter remained relatively constant at 70-200 nmol/ml. There was no difference among foremilk, middle milk and hind milk, nor was there a diurnal pattern of variation in unesterified choline concentrations. Milk phosphatidylcholine and sphingomyelin concentrations remained relatively constant throughout lactation (100-200 nmol/ml). Hind milk always contained more of these phospholipids than did foremilk or middle milk. There was no consistent diurnal pattern of variation in milk concentrations of phosphatidylcholine or sphingomyelin. Milk contained no phospholipase activity capable of forming free choline from phosphatidylcholine or sphingomyelin. Bovine milk contained approximately the same concentrations of choline, phosphatidylcholine and sphingomyelin as did human milk from mothers more than 15 d postpartum. The same was true of "humanized" infant formulas made from cow's milk. Soy protein-based formulas had much more unesterified choline (up to 650 nmol/ml) and much less sphingomyelin than did mature human milk.


Perspectives of staff nurses of the reasons for and the nature of patient-initiated call lights: an exploratory survey study in four USA hospitals.
Little research has been done on patient call light use and staff response time, which were found to be associated with inpatient falls and satisfaction. Nurses' perspectives may moderate or mediate the aforementioned relationships. This exploratory study intended to understand staff's perspectives about call lights, staff responsiveness, and the reasons for and the nature of call light use. It also explored differences among hospitals and identified significant predictors of the nature of call light use. This cross-sectional, multihospital survey study was conducted from September 2008 to January 2009 in four hospitals located in the Midwestern region of the United States. A brief survey was used. All 2309 licensed and unlicensed nursing staff members who provide direct patient care in 27 adult care units were invited to participate. A total of 808 completed surveys were retrieved for an overall response rate of 35%. The SPSS 16.0 Window version was used. Descriptive and binary logistic regression analyses were conducted. The primary reasons for patient-initiated calls were for toileting assistance, pain medication, and intravenous problems. Toileting assistance was the leading reason. Each staff responded to 6 to 7 calls per hour and a call was answered within 4 minutes (estimated). 49% of staff perceived that patient-initiated calls mattered to patient safety. 77% agreed that that these calls were meaningful. 52% thought that these calls required the attention of nursing staff. 53% thought that answering calls prevented them from doing the critical aspects of their role. Staff's perceptions about the nature of calls varied across hospitals. Junior staff tended to overlook the importance of answering calls. A nurse participant tended to perceive calls as more likely requiring nursing staff's attention than a nurse aide participant. If answering calls was a high priority among nursing tasks, staff would perceive calls as being important, requiring nursing staff's attention, and being meaningful. Therefore, answering calls should not be perceived as preventing staff from doing the critical aspects of their role. Additional efforts are necessary to reach the ideal or even a reasonable level of patient safety-first practice in current hospital environments.


[Structure of the blood microcirculatory bed in intramural uterine myomas].
Histological, morphometric and histochemical examination of uterine samples was carried out in 33 cases of intramural myoma (aged 26-45) and 32 autopsied controls with unaltered uterus (20-45 years). Peripheral areas of myomas showed a polymeric-homonomous structure of the blood microcirculatory bed. However, it was characterized by higher specific volumes of all components as compared to controls. This, perhaps, may account for local circulatory hyperestrogenism which supports myoma progression. Pronounced changes in the structure of microvessels of the central areas were matched by a 2-4-fold decrease in their specific volumes. The degree of said alteration in other areas of the myometrium was found to be in direct correlation with the size of myoma.


Influence of Initial Residual Stress on Growth and Pattern Creation for a Layered Aorta.
Residual stress is ubiquitous and indispensable in most biological and artificial materials, where it sustains and optimizes many biological and functional mechanisms. The theory of volume growth, starting from a stress-free initial state, is widely used to explain the creation and evolution of growth-induced residual stress and the resulting changes in shape, and to model how growing bio-tissues such as arteries and solid tumors develop a strategy of pattern creation according to geometrical and material parameters. This modelling provides promising avenues for designing and directing some appropriate morphology of a given tissue or organ and achieve some targeted biomedical function. In this paper, we rely on a modified, augmented theory to reveal how we can obtain growth-induced residual stress and pattern evolution of a layered artery by starting from an existing, non-zero initial residual stress state. We use experimentally determined residual stress distributions of aged bi-layered human aortas and quantify their influence by a magnitude factor. Our results show that initial residual stress has a more significant impact on residual stress accumulation and the subsequent evolution of patterns than geometry and material parameters. Additionally, we provide an essential explanation for growth-induced patterns driven by differential growth coupled to an initial residual stress. Finally, we show that initial residual stress is a readily available way to control growth-induced pattern creation for tissues and thus may provide a promising inspiration for biomedical engineering.


Updates in the management of sinonasal mucosal melanoma.
Sinonasal mucosal melanoma (SNMM) is an aggressive cancer with a poor prognosis. Although there is significant study surrounding the treatment of sinonasal malignancies and cutaneous melanomas, the rarity of this tumor has largely precluded robust outcomes analyses. The authors of this review seek to provide an overview of the recent literature related to the treatment of SNMM with added context from our institutional experience with this disease. In the surgical management of sinonasal malignancies and SNMM specifically, resection via endoscopic endonasal technique appears to offer comparable oncologic outcomes versus an open approach. The role of adjuvant therapy continues to be debated, but there is strong evidence for improved rates of local control with radiotherapy after complete resection. In the last few years, significant developments have been made in the study of systemic therapies for cutaneous melanoma. The identification of genetic mutations common to mucosal melanoma has allowed for early trials of targeted therapies, but study is ongoing. Although the study of SNMM is largely limited to small retrospective case series, treatment continues to evolve. Until effective systemic therapies can be identified, endoscopic resection with adjuvant radiotherapy may offer the best disease-free survival with acceptably low morbidity.


Universal EDXRF Method for Multi-elemental Determinations Using Fused Bead Specimens.
A systematic study on the EDXRF determination of multi-elements using fusion bead specimens and a universal calibration procedure was carried out. Beads of multi-elements were prepared by mixing MERCK multi-elemental standard solutions with the fusion flux. The elemental X-ray lines of K, Ca, Ba, Cr, Mn, Fe, Co, Ni, Cu and Zn were excited using a Ge secondary target and that of Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Pb, Bi and Sr using a Rh primary target. The relative sensitivities of the elemental X-ray lines obtained using different excitation modes were calculated using a single bead. Using this relative sensitivity, the concentration of elements in other bead samples was determined. The average precision obtained was 3% (1 s) and the average deviation from expected values, calculated on the basis of sample preparation, was 6%. In order to further countercheck the validity of this methodology, a standard soil sample received from IAEA was analyzed.


The human brain from above: an increase in complexity from environmental stimuli to abstractions.
Contrary to common belief, the brain appears to increase the complexity from the perceived object to the idea of it. Topological models predict indeed that: (a) increases in anatomical/functional dimensions and symmetries occur in the transition from the environment to the higher activities of the brain, and (b) informational entropy in the primary sensory areas is lower than in the higher associative ones. To demonstrate this novel hypothesis, we introduce a straightforward approach to measuring island information levels in fMRI neuroimages, via Rényi entropy derived from tessellated fMRI images. This approach facilitates objective detection of entropy and corresponding information levels in zones of fMRI images generally not taken into account. We found that the Rényi entropy is higher in associative cortices than in the visual primary ones. This suggests that the brain lies in dimensions higher than the environment and that it does not concentrate, but rather dilutes messages coming from external inputs.


Fungi of quarantine concern for China I: Dothideomycetes.
The current list of Chinese quarantine pests includes 130 fungal species. However, recent changes in the taxonomy of fungi following the one fungus = one name initiative and the implementation of DNA phylogeny in taxonomic revisions, resulted in many changes of these species names, necessitating an update of the current list. In addition, many quarantine fungi lack modern morphological descriptions and authentic DNA sequences, posing significant challenges for the development of diagnostic protocols. The aim of the present study was to review the taxonomy and names of the 33 Chinese quarantine fungi in Dothideomycetes, and provide reliable DNA barcodes to facilitate rapid identification. Of these, 23 names were updated according to the single name nomenclature system, including one new combination, namely Cophinforma tumefaciens comb. nov. (syn. Sphaeropsis tumefaciens). On the basis of phylogenetic analyses and morphological comparisons, a new genus Xenosphaeropsis is introduced to accommodate the monotypic species Xenosphaeropsis pyriputrescens comb. nov. (syn. Sphaeropsis pyriputrescens), the causal agent of a post-harvest disease of pears. Furthermore, four lectotypes (Ascochyta petroselini, Mycosphaerella ligulicola, Physalospora laricina, Sphaeria lingam), three epitypes (Ascochyta petroselini, Phoma lycopersici, Sphaeria lingam), and two neotypes (Ascochyta pinodella, Deuterophoma tracheiphila) are designated to stabilise the use of these names. A further four reference strains are introduced for Cophinforma tumefaciens, Helminthosporium solani, Mycocentrospora acerina, and Septoria linicola. In addition, to assist future studies on these important pathogens, we sequenced and assembled whole genomes for 17 species, including Alternaria triticina, Boeremia foveata, B. lycopersici, Cladosporium cucumerinum, Didymella glomerata, Didymella pinodella, Diplodia mutila, Helminthosporium solani, Mycocentrospora acerina, Neofusicoccum laricinum, Parastagonospora pseudonodorum, Plenodomus libanotidis, Plenodomus lingam, Plenodomus tracheiphilus, Septoria petroselini, Stagonosporopsis chrysanthemi, and Xenosphaeropsis pyriputrescens. Citation: Zhao P, Crous PW, Hou LW, et al. 2021. Fungi of quarantine concern for China I: Dothideomycetes. Persoonia 47: 45-105. https://doi.org/10.3767/persoonia.2021.47.02.


Tolerance induction in composite facial allograft transplantation in the rat model.
Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.


Estrogen ameliorates microglial activation by inhibiting the Kir2.1 inward-rectifier K(+) channel.
Microglial activation is implicated in the pathogenesis of Parkinson's disease (PD). Although the etiology of PD remains unclear, age and male gender are known PD risk factors. By comparing microglia and dopaminergic (DA) neurons in the substantia nigra (SN) of male and female mice of different ages, we found that the degrees of microglial activation and DA neuron loss increased with age in both genders, but were more pronounced in males, as were peripheral lipopolysaccharide (LPS)-induced microglial activation and DA neuron loss. A bilateral ovariectomy (OVX) eliminated the female-associated protection against age- and LPS-induced microglial activation, which suggests that ovary hormones are involved in gender-specific responses. Treating female mice with 17β-estradiol supplements reduced the age-associated microglial activation in OVX mice. Moreover, pretreating mouse BV2 microglial cells with 17β-estradiol inhibited LPS-induced elevation of Toll-like receptor 4, phosphorylated p38, and TNF-α levels. We then examined the effect of 17β-estradiol on inward-rectifier K(+) channel Kir2.1, a known regulator of microglial activation. We found that 17β-estradiol inhibited the Kir2.1 activity of BV2 cells by reducing the probability that the channel would be open. We conclude that age- and inflammation-associated microglial activation is attenuated by ovarian estrogen, because it inhibits Kir2.1.


PARP inhibitors and epithelial ovarian cancer: Molecular mechanisms, clinical development and future prospective.
Epithelial ovarian cancer (EOC) has a poor prognosis. Since the introduction of paclitaxel as antineoplastic agent >20 years ago, only a few phase III randomized trials have shown challenging data regarding different therapeutic options for facing its aggressive clinical course and granting active therapies to patients. Different studies have shown the utility of poly(ADP-ribose) polymerase (PARP) inhibitors in women with EOC with or without BRCA mutations, both germline and somatic. Three PARP inhibitors, olaparib, rucaparib and niraparib, have been recently approved by the Food and Drug Administration for clinical use in EOC patients, though with different clinical indications and profiles of toxicity, while two other molecules, veliparib and talazoparib, are still under clinical investigation. The aim of the present paper is to evaluate the current status of PARP inhibitors in terms of molecular activity, pharmacodynamic properties and clinical applications.


Events from ovulation to implantation studied by three-dimensional ultrasound.
During the last decade transvaginal color Doppler has played an important role in increasing understanding of early human development. Although our knowledge of early pregnancy development has recently improved, little is known about the most critical period of human development: between conception and implantation. Recent advances in 3D ultrasound have made studies of follicular development, ovulation and uterine receptivity more accurate. The same method can be used for evaluation of the Fallopian tube patency and assessment of the ovarian and uterine causes of infertility which hamper processes of early human development. Storage capacities, reconstruction of the volume images and simultaneous viewing of all three orthogonal planes are the main advantages of this method. Introducing 3D ultrasound into assessment of early pregnancy has enabled visualization and volume estimation of the gestational sac, yolk sac and embryo. Switching on power Doppler superimposed to 3D gray scale can detect early vasculogenesis within the intervillous space and embryo/fetus. Here we review the potential application of this novel technique in monitoring the morphological and functional processes from ovulation towards implantation and early pregnancy.


Different morphometric and densitometric parameters predict cervical and trochanteric hip fracture: the EPIDOS Study.
We used an experimental software measuring the hip axis length (HAL) and bone mineral density (BMD) in specific regions of the lower and upper part of the femoral neck on dual-energy X-ray absorptiometry scans. To determine whether these parameters were significant predictors of the type of hip fracture, we measured 167 healthy women (controls), 24 women with trochanteric, and 42 women with cervical hip fractures within the EPIDOS prospective cohort. EPIDOS is a multicenter prospective study on risk factors for hip fracture performed in 7575 elderly women living at home, aged 75-95 and conducted in five French centers (Amiens, Lyon, Montpellier, Paris, Toulouse). Measurements were performed on data acquired at baseline before the occurrence of fracture. In the cervical fracture group, HAL was significantly longer than in controls (94.2 vs. 92.3, p = 0.03), and the associated odds ratio (OR) adjusted for age, weight, and total femoral neck BMD was significant (OR = 1.64, 95% confidence interval [CI] 1.06-2.55). In contrast, HAL was not significantly different from controls in the trochanteric fracture group. Femoral neck diameter was not a predictor of fracture. The upper and lower femoral neck BMD was lower in the trochanteric fracture group than in controls, and both measurements predicted trochanteric femoral neck fracture. In contrast, the prediction of cervical femoral neck fracture was enhanced by measuring only the upper part of the femoral neck (OR = 2.79 vs. 1.97 for the total femoral neck) while BMD of the lower part was not different from controls. Hip axis length is a predictor of femoral neck fracture. Femoral neck BMD distribution is different between cervical and trochanteric fractures. These results support the hypothesis of a different pathophysiological mechanism between the two types of hip fractures.


Effects of hospital safety-net burden and hospital volume on failure to rescue after open abdominal aortic surgery.
Failure to rescue (FTR) is defined as the inability to rescue a patient from major perioperative complications, resulting in operative mortality. FTR is a known contributor to operative mortality after open abdominal aortic surgery. Understanding the causes of FTR is essential to designing interventions to improve perioperative outcomes. The objective of this study was to determine the relative contributions of hospital volume and safety-net burden (the proportion of uninsured and Medicaid-insured patients) to FTR. The Nationwide Inpatient Sample (2001-2011) was analyzed to investigate variables associated with FTR after elective open abdominal aortic operations in the United States. FTR was defined as in-hospital death following postoperative complications. Mixed multivariate regression models were used to assess independent predictors of FTR, taking into account the clustered structure of the data (patients nested into hospitals). A total of 47,233 elective open abdominal aortic operations were performed in 1777 hospitals during the study period. The overall incidences of postoperative complications, in-hospital mortality, and FTR in the whole cohort were 32.7%, 3.2%, and 8.6%, respectively. After adjusting for demographics, comorbidities, and hospital characteristics, safety-net burden was significantly associated with increased likelihood of FTR (highest vs lowest quartile of safety-net burden, odds ratio, 1.59; 95% confidence interval, 1.32-1.91; P < .0001). In contrast, after adjusting for safety-net burden, procedure-specific hospital volume was not significantly associated with FTR (P = .897). After adjusting for patient- and hospital-level variables, including hospital volume, safety-net burden was an independent predictor of FTR after open aortic surgery. Future investigations should be aimed at better understanding the relationship between safety-net hospital burden and FTR to design interventions to improve outcomes after open abdominal aortic surgery.


Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis.
Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related secreted proteins that limit matrix metalloproteinase (MMP) activity and also have direct effects on cell growth. We used the highly efficient adenoviral delivery system to overexpress individual TIMPs from the cytomegalovirus immediate early promoter in rat aortic smooth muscle cells. Overexpression of TIMP-1, -2, or -3, or a synthetic MMP inhibitor similarly inhibited SMC chemotaxis and invasion through reconstituted basement membrane. TIMP-1 overexpression did not effect cell proliferation. By contrast, TIMP-2 caused a dose-dependent reduction in proliferation, an effect not mimicked by a synthetic MMP inhibitor. TIMP-3 overexpression induced DNA synthesis, and promoted SMC death by apoptosis, a phenotype reproduced by adding TIMP-3 to uninfected cells, but not by a synthetic MMP inhibitor. Our study is the first to compare systematically the effect of overexpression of three TIMPs in any cell. We found similar effects on invasion mediated by inhibition of MMP activity, but widely divergent effects on proliferation and death through actions of TIMP-2 and -3 independent of MMP inhibition. These findings have important implications for the physiological roles of TIMPs and their use in gene therapy.


Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic.
Immunization with Haemophilus influenzae type b (Hib) conjugate polysaccharide vaccines has dramatically reduced Hib disease worldwide. As in other populations, nasopharyngeal carriage of Hib declined markedly in Aboriginal infants following vaccination, although carriage has not been entirely eliminated. In this study, we describe the genetic characteristics and the carriage dynamics of longitudinal isolates of Hib, characterized by using several typing methods. In addition, carriage rates of nonencapsulated H. influenzae (NCHi) are high, and concurrent colonization with Hib and NCHi is common; we also observed NCHi isolates which were genetically similar to Hib. There is a continuing need to promote Hib immunization and monitor H. influenzae carriage in populations in which the organism is highly endemic, not least because of the possibility of genetic exchange between Hib and NCHi strains in such populations.


Functions of intermediate filaments in neuronal development and disease.
Five major types of intermediate filament (IF) proteins are expressed in mature neurons: the three neurofilament proteins (NF-L, NF-M, and NF-H), alpha-internexin, and peripherin. While the differential expression of IF genes during embryonic development suggests potential functions of these proteins in axogenesis, none of the IF gene knockout experiments in mice caused gross developmental defects of the nervous system. Yet, deficiencies in neuronal IF proteins are not completely innocuous. Substantial developmental loss of motor axons was detected in mice lacking NF-L and in double knockout NF-M;NF-H mice, supporting the view of a role for IFs in axon stabilization. Moreover, the absence of peripherin resulted in approximately 30% loss of small sensory axons. Mice lacking NF-L had a scarcity of IF structures and exhibited a severe axonal hypotrophy, causing up to 50% reduction in conduction velocity, a feature that would be very detrimental for large animal species. Unexpectedly, the NF-M rather than NF-H protein turned out to be required for proper radial growth of large myelinated axons. Studies with transgenic mice suggest that some types of IF accumulations, reminiscent of those found in amyotrophic lateral sclerosis (ALS), can have deleterious effects and even cause neurodegeneration. Additional evidence for the involvement of IFs in pathogenesis came from the recent discovery of neurofilament gene mutations linked to ALS and Charcot-Marie-Tooth disease (CMT2E). Conversely, we discuss how certain types of perikaryal neurofilament aggregates might confer protection in motor neuron disease.


Pseudomonas putrefaciens and group A beta-hemolytic streptococcus septicemia.
Pseudomonas putrefaciens was isolated twice in blood cultures from which group A beta-hemolytic streptococci were concurrently isolated from a moribund woman. Only once previously has P putrefaciens bacteremia been reported. To our knowledge, we are reporting the first blood isolate of P putrefaciens, albeit with group A beta-hemolytic streptococci, associated with fatal septicemia.


Utility of hydrogen breath tests in diagnosis of small intestinal bacterial overgrowth in malabsorption syndrome and its relationship with oro-cecal transit time.
Small intestinal bacterial overgrowth (SIBO), which may result from intestinal stasis, is common in malabsorption syndrome (MAS). Quantitative culture of upper gut aspirate is used as a gold standard for the diagnosis of SIBO. Studies on diagnosis of SIBO using non-invasive hydrogen breath tests are contradictory. 83 patients (age 35 [14-70] y; 50 men) with MAS due to various causes were investigated for SIBO using quantitative culture of upper gut aspirate obtained using a special endoscopic catheter and glucose and lactulose hydrogen breath tests (GHBT, LHBT). Sustained elevation in breath hydrogen of 12 ppm above basal and two separate peaks (one due to SIBO and the other from colon) were diagnostic of SIBO in GHBT and LHBT, respectively. Oro-cecal transit time (OCTT) was estimated using LHBT in 71 patients. Thirty two of 81 (39.5%) patients with MAS had SIBO on culture (>or= 10(5) CFU/mL). Using aspirate culture as the gold standard, sensitivity, specificity, positive and negative predictive values and diagnostic accuracy of GHBT to diagnose SIBO were 44%, 80%, 62%, 67% and 65%, respectively; the corresponding values for LHBT were 31%, 86%, 62%, 54% and 55%, respectively. OCTT in patients with SIBO diagnosed on GHBT and/or aspirate culture (n=58) was longer than in those without (170 [60-250] vs. 120 [50-290] min, p=0.02); of others, 7 were hydrogen non-producers and in 6 OCTT could not be assessed due to sustained early peak because of SIBO. GHBT and LHBT are highly specific but insensitive for diagnosis of SIBO in MAS; OCTT is longer in patients with MAS and SIBO than in those without.


The Modular Concept in Skull Base Surgery: Anatomical Basis of the Median, Paramedian and Lateral Corridors.
Introduction A thorough understanding of skull base anatomy is imperative to perform safely and effectively any skull base approach. In this article, we examine the microsurgical anatomy of the skull base by proposing a modular topographic organization in the median, paramedian, and lateral surgical corridors in relation to transcranial and endoscopic approaches. Methods Five dry skulls were studied focusing on the intracranial and exocranial skull base. Two lines were drawn parallel to the lateral border of the cribriform plate of the ethmoid bone and foramen lacerum, respectively. Lines 1 and 2 delimited the median, paramedian and lateral corridors of the skull base. The bony structures that formed each corridor were carefully reviewed in relation to the planning and execution of the skull base transcranial and endoscopic approaches. Results The midline corridor involves the crista galli, cribriform plate, planum and jugum sphenoidale, chiasmatic sulcus, tuberculum sellae, sellar region, dorsum sellae, clivus, and foramen magnum. The paramedian corridor includes the fovea ethmoidalis, the root of the lesser and greater sphenoid wing, anterior clinoid process, foramen lacerum, the upper half of the petro-occipital suture, and jugular tubercle. The lateral corridors include the orbital plates, sphenoid wings, squamosal and petrous parts of the temporal bone, caudal aspect of the petro-occipital suture, internal auditory canal, jugular foramen, the sulcus of the sigmoid sinus. Conclusion In-depth three-dimensional knowledge of skull base anatomy based on the modular concept of the surgical corridors is critical for the planning and execution of the transcranial and endoscopic approaches.


Suppression of Apoptosis in Human Umbilical Vein Endothelial Cells (HUVECs) by Klotho Protein is Associated with Reduced Endoplasmic Reticulum Oxidative Stress and Activation of the PI3K/AKT Pathway.
BACKGROUND Klotho protein has been shown to act as a hormone on the cardiovascular system, and to have specific protective effects on vascular endothelial cells. The aim of this study was to investigate the mechanisms of the anti-oxidative and anti-apoptotic effects of klotho protein on hydrogen peroxide (H₂O₂)-induced apoptosis and endoplasmic reticulum oxidative stress in human umbilical vein endothelial cells (HUVECs). MATERIAL AND METHODS HUVECs were cultured in vitro and treated with H₂O₂. The MTT assay evaluated cell viability of H₂O₂-treated HUVECs, and flow cytometry measured cell apoptosis. An enzyme-linked immunosorbent assay (ELISA) measured the levels of nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-6. Western blot was used to detect the expression of the proteins, 78 kD glucose-regulated protein (GRP78), CCAAT-enhancer-binding protein homologous protein (CHOP), caspase-3, caspase-9, caspase-12, and AKT. The effects of LY294002, a pharmacological inhibitor of PI3K, were evaluated. RESULTS Klotho protein increased the viability of H2O2-treated HUVECs and reduced the expression of NO, TNF-α, and IL-6. Klotho protein reduced the rate of apoptosis of H₂O₂-treated HUVECs and downregulated the expression of proteins associated with endoplasmic reticulum oxidative stress, GRP78 and CHOP, and the expression of the apoptotic proteins, caspase-3, caspase-9, and caspase-12, and activated the phosphorylation of AKT. The addition of LY294002 inhibited klotho protein downregulation of GRP78, CHOP, caspase-3, caspase-9, and caspase-12 expression. CONCLUSIONS In HUVECs, klotho protein suppressed apoptosis mediated by endoplasmic reticulum oxidative stress by activation of the PI3K/AKT pathway.


A comparative approach to characterize the landscape of host-pathogen protein-protein interactions.
Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors. This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells. A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.


Setting reference values for exhaled nitric oxide: a systematic review.
The values obtained when the fraction of exhaled nitric oxide (FeNO) is measured are affected by several factors that are specific to the individual patient, making interpretation difficult, especially in the initial assessment of patients with respiratory symptoms. Systematic review of studies on FeNO reference values and individual-specific factors that influence them. From 3739 references, 15 studies were included. Four studies included children and adolescents. In nine studies, samples were selected from the general population. Most studies reported objective measures for atopy (nine studies), but not for smoking status (one). Significant determinants of FeNO values reported were age and height (seven studies), atopy (six), smoking (four), weight (four), sex (three) and race (three). Additional factors were included in eight studies. R2 was reported in only five studies. The logarithmic transformation of FeNO was inadequately described in seven studies. There are several equations for FeNO reference values that may be used in clinical practice, although the factors they include and the statistical methods they use vary considerably. We recommend the development of standard methods for the evaluation of normal FeNO data and that reference equations should be formulated based on a predetermined physiological model.


[The medical rationale category and a new epistemology in health].
This article is an analytical report on the 20-year trajectory of the 'medical rationale' category that emerged in the early 1990s in the area of Social and Human Sciences in Health in the field of Public Health. Its objective was to study complex and therapeutic medical systems and traditional, complementary and alternative medicines. Based on a critical review of the literature, it presents some aspects of the cultural, political, institutional and social context of its emergence, as well as its main contributions and developments on a theoretical level and on social policies and practices in health. The southern epistemology concept of Boaventura de Sousa Santos is used to reflect upon the contribution of the 'medical rationale' category to the critique of the post-modern scientific rationale and to the creation of a new epistemology in health.


Nanoparticles containing anti-inflammatory agents as chemotherapy adjuvants: optimization and in vitro characterization.
The pre-administration of dexamethasone (DEX) has previously been shown to enhance the anti-tumor efficacy of chemotherapeutic agents. The delivery of anti-inflammatory agents specifically to tumors via nanoparticle carriers is expected to promote the effectiveness of chemotherapeutic agents while avoiding systemic toxicities. The process for preparing solid lipid nanoparticles containing anti-inflammatory agents using the nanotemplate engineering method was optimized. Due to the solubilization of DEX in the bulk aqueous phase, its more lipophilic palmitate ester was synthesized and incorporated in nanoparticles that included a pegylating agent, PEG6000 mono-stearate, as part of the formulation. The stealth properties of these nanoparticles were demonstrated to be enhanced compared to latex particles by measuring the adsorption of radioiodinated IgG (185 microg vs. 6.7 microg IgG/mg NP). In addition, the uptake of (14)C-labeled nanoparticles by murine macrophages was shown to decrease from 36.6% to 14.7% of the nanoparticles/mg cell protein as the amount of pegylating agent in the formulation increased from 0 to 4 mg/mL. The high loading values and low burst effect observed for these DEX palmitate-containing nanoparticles in addition to their stealth properties are expected to allow for the delivery of sufficient amounts of DEX to tumors to enhance the uptake of chemotherapeutic agents.


Pro-IL-1β Is an Early Prognostic Indicator of Severe Donor Lung Injury During Ex Vivo Lung Perfusion.
Ex vivo lung perfusion (EVLP) is used to evaluate and recondition extended criteria donor lungs for transplantation. Interleukin-1β (IL-1β) has been identified as a prognostic indicator of nonrecovery during EVLP. This may be an effect of inflammasome activation or cellular necrosis following donation and graft preservation. Delineating the mechanism of IL-1β release is required. The inactive intracellular precursor molecule, pro-IL-1β, was characterized along with the pro-IL-1β processing enzyme, caspase-1, in the perfusate of n = 20 human lungs that had undergone EVLP (n = 10 lungs that failed to recover and were discarded versus n = 10 lungs that reconditioned and were transplanted). In an experimental porcine model, n = 8 lungs underwent EVLP and were randomized to receive either a specific NLRP3 inflammasome inhibitor or control. Significant increases in pro-IL-1β and caspase-1 were observed in the perfusate from human lungs that did not recondition during EVLP compared with those that successfully reconditioned and were used for transplantation. Within the porcine EVLP, NLRP3 inflammasome inhibition reduced IL-1β within the perfusate compared with controls, but this had no impact on lung function, hemodynamics, or inflammation. Our data suggest that pro-IL-1β is passively released following cellular necrosis of the donor lung.


Frequency and predictive factors of malignancy in residual thyroid tissue and cervical lymph nodes after partial thyroidectomy for differentiated thyroid cancer.
The extent of surgery in differentiated thyroid cancer (DTC) has been a controversial issue. Total thyroidectomy potentially carries a higher operative risk, whereas partial thyroidectomy has the risk of leaving significant residual malignancy. The aim of this study was to assess the frequency and potential predictive factors of malignancy in the residual thyroid tissue and the cervical lymph nodes (CLN) in patients with DTC who had partial thyroid surgery and subsequently underwent completion thyroidectomy and/or modified neck dissection. Age, gender, pressure symptoms, duration of symptoms, size of the original tumor, tumor multifocality, perithyroidal tumor extension, soft tissue invasion, and serum thyroglobulin (Tg) level after first surgery were analyzed as potential predictive factors for the presence of malignancy in the thyroid remnant and the CLN. We retrospectively reviewed the medical and pathologic data of 101 cases of DTC; 97 had papillary and 4 had follicular thyroid cancer. On the initial surgery, the median tumor size was 2.5 cm (range, 0.5 to 8.5 cm). Tumor multifocality occurred in 28 cases, perithyroidal tumor extension in 26 cases, and soft tissue invasion in 9 cases. Completion thyroidectomy was performed in 100 cases and modified neck dissection in 90 cases. On completion neck surgery, 39 patients had evidence of malignancy in the residual thyroid tissue and 36 patients in the CLN. In 23 (22.7%) cases, malignancy was present in both CLN and residual thyroid tissue. Only tumor multifocality and Tg level greater than 20 ng/mL after first surgery were predictive of the presence of malignancy in the thyroid remnant, whereas age older than 40 years, soft tissue invasion, perithyroidal tumor extension, and Tg level greater than 20 ng/mL were predictive of malignancy in CLN. Residual malignancy is common after partial thyroid surgery for DTC. Tumor multifocality and Tg level may be predictive of its presence in residual thyroid tissue. Age, perithyroidal tumor extension, soft tissue invasion, and Tg level are predictive of the presence of lymph node metastases.


Development linear and non-linear QSAR models for predicting AXL kinase inhibitory activity of N-[4-(quinolin-4-yloxy)phenyl]benzenesulfonamides.
In this research, we used CoMFA, LSSVM and FFANN for creating QSAR models for predicting AXL Kinase inhibitory activity of N-[4-(Quinolin-4-yloxy)phenyl]benzenesulfonamides. A CoMFA model with three components was developed and CoMFA contour maps were interpreted to extract chemical features that influence the inhibitory activity of these molecules. R2 for train and test set of CoMFA model were 0.8900 and 0.8171, respectively. Model created by five Dragon descriptors and LSSVM model showed slightly better predictive power with respect to CoMFA model. R2 for train, test set of created LSSVM model were 0.0.8477 and 0.8218, respectively. Also, a FFANN model, using the same five descriptors, was developed with 2 neurons in its hidden layer and R2 for its train and test sets were 0.8314 and 0.8522, respectively. All created models were validated by calculating several statistical parameters and their applicability domain were investigated by calculating leverage. Furthermore, a homology model was built for Axl structure and molecules with the lowest and the greatest activity were docked to it and their interactions with Axl were investigated.


Identification of the molecular attributes required for aminoglycoside activity against Leishmania.
Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.


Highly active, lipase silicone elastomers.
Lipase Candida rugosa was entrapped in silicone rubber via condensation-cure room temperature vulcanization of silanol-terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, to give a highly active silicone-enzyme elastomer. The effect on enzyme activity of addition of water and hydrophilic polymeric moieties based on poly(ethylene oxide) 2 was examined, as were crosslinker concentration, enzyme concentration, and elastomer thickness. It was demonstrated that lipase is most active in silicone elastomers and more active in silicone oils than simple hydrocarbons. Crosslink density in these elastomers was not an important factor in the reactivity of the rubber. However, the addition of hydrophilic species prior to elastomer formation decreased the efficiency both of the dispersion of the enzyme and the resulting activity of the elastomer. This effect could be moderated by prior exposure of the lipase to silicone oil. Thus, hydrophobic silicones play a protective/activating role for lipase.


Epidermal growth factor receptor inhibition reduces angiogenesis via hypoxia-inducible factor-1α and Notch1 in head neck squamous cell carcinoma.
Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.


Compact direct space-to-time pulse shaping with a phase-only spatial light modulator.
A very compact and innovative pulse shaper is proposed and demonstrated. The standard architecture for pulse shaping that is composed of diffraction gratings associated with an amplitude-phase spatial light modulator (SLM) is replaced by a single phase-only SLM. It acts as a pulse stretcher and as an amplitude and phase modulator at the same time. Preliminary experiments demonstrate the accurate control of amplitude and phase of shaped pulses.


[Shifts in the heart left ventricle function in patients with pulmonary tuberculosis during adequate chemotherapy].
To investigate the hemodynamics and myocardic contraction of the heart left ventricle, 61 patients with pulmonary tuberculosis (main group) and 26 healthy subjects (control group) were observed. Higher ultimate systolic and diasystolic volumes of the left ventricle and lower levels of the efflux fraction in the patients with active pulmonary tuberculosis were stated. There was shown inverse correlation of the systemic systolic arterial pressure and the left ventricle efflux fraction with ESR, evident of the tuberculosis intoxication. The most pronounced aggravation of the left ventricle function was recorded in the patients with the most severe tuberculosis process. The impairments in the left ventricle in the patients with active pulmonary tuberculosis were of functional nature. Due to intensive therapy of the tuberculosis, the indices of the left ventricle efflux function improved and the systemic arterial pressure came to normal, along with elimination of the tuberculosis intoxication signs.


Genetic signature of amphimixis allows for the detection and fine scale localization of sexual reproduction events in a mainly parthenogenetic nematode.
Asexuality is an important mode of reproduction in eukaryotic taxa and has a theoretical advantage over sexual reproduction because of the increased ability to propagate genes. Despite this advantage, hidden signs of cryptic sex have been discovered in the genomes of asexual organisms. This has provided an interesting way to address the evolutionary impact of sex in plant and animal populations. However, the identification of rare sexual reproduction events in mainly asexual species has remained a challenging task. We examined the reproductive history in populations of the plant parasitic nematode Xiphinema index by genotyping individuals collected from six grapevine fields using seven microsatellite markers. A high level of linkage disequilibrium and heterozygous excess suggested a clonality rate of 95-100%. However, we also detected rare sexual reproduction events within these highly clonal populations. By combining highly polymorphic markers with an appropriate hierarchical sampling, and using both Bayesian and multivariate analysis with phylogenetic reconstructions, we were able to identify a small number of sexually produced individuals at the overlapping zones between different genetic clusters. This suggested that sexual reproduction was favoured when and where two nematode patches came into contact. Among fields, a high degree of genetic differentiation indicated a low level of gene flow between populations. Rare genotypes that were shared by several populations suggested passive dispersal by human activities, possibly through the introduction of infected plants from nurseries. We conclude that our method can be used to detect and locate sexual events in various predominantly asexual species.


[The application of improved Glasgow coma scale score of 15 as switching point for invasive noninvasive mechanical ventilation in treatment of severe respiratory failure in chronic obstructive pulmonary disease].
To estimate the feasibility and the efficacy of early extubation and the sequential non invasive mechanical ventilation (MV) in severe respiratory failure of chronic obstructive pulmonary disease (COPD) with the improved Glasgow coma scale (GCS) score of 15 as the switching point. By a prospective control study, 20 patients with COPD and respiratory failure who had undergone endotracheal intubation and MV from March 2007 to November 2009 were enrolled as treatment group. Invasive MV with synchronous intermittent mandatory ventilation and pressure support ventilation (SIMV+PSV) pattern were given to these patients. When the period of "improved GCS score of 15 standard" window period appeared and being kept for 2 hours, endotracheal tube was extubated, and nasal mask with PSV+positive end expiratory pressure (PEEP) was used, followed by gradual decrease of the level of pressure support till weaning of MV. Nineteen patients who were treated with MV with ordinary way of weaning from March 2005 to March 2007 served as the control group. Prior to the MV, the ventilation and oxygenation index , the length of invasive MV, total MV time, total hospital stay, re intubation and ventilator associated pneumonia (VAP) occurred in the number of cases were observed and compared between two groups. There was no significant difference in the ventilation and oxygenation index prior to the MV. Compared with control group, in treatment group, the length of invasive ventilation (days: 3.2±1.1 vs. 10.5±3.2), the total duration of MV (days: 4.8±2.5 vs. 10.5±3.2), the length of hospital stay (days: 17±3 vs. 22±7) were significantly shorter (all P<0.01), and the incidence of VAP was significantly lower (cases: 0 vs. 5, P<0.01), while the number of re intubation was slightly higher but without statistical significance (cases: 3 vs. 1, P>0.05). The application of improved GCS score of 15 as the switching point with 2 hours as window period for early extubation and non invasive nasal mask ventilation can significantly improve the therapeutic effect in patients with severe respiratory failure in COPD.


Accuracy of respiratory gas variables, substrate, and energy use from 15 CPET systems during simulated and human exercise.
Various systems are available for cardiopulmonary exercise testing (CPET), but their accuracy remains largely unexplored. We evaluate the accuracy of 15 popular CPET systems to assess respiratory variables, substrate use, and energy expenditure during simulated exercise. Cross-comparisons were also performed during human cycling experiments (i.e., verification of simulation findings), and between-session reliability was assessed for a subset of systems. A metabolic simulator was used to simulate breath-by-breath gas exchange, and the values measured by each system (minute ventilation [V̇E], breathing frequency [BF], oxygen uptake [V̇O2 ], carbon dioxide production [V̇CO2 ], respiratory exchange ratio [RER], energy from carbs and fats, and total energy expenditure) were compared to the simulated values to assess the accuracy. The following manufacturers (system) were assessed: COSMED (Quark CPET, K5), Cortex (MetaLyzer 3B, MetaMax 3B), Vyaire (Vyntus CPX, Oxycon Pro), Maastricht Instruments (Omnical), MGC Diagnostics (Ergocard Clinical, Ergocard Pro, Ultima), Ganshorn/Schiller (PowerCube Ergo), Geratherm (Ergostik), VO2master (VO2masterPro), PNOĒ (PNOĒ), and Calibre Biometrics (Calibre). Absolute percentage errors during the simulations ranged from 1.15%-50.3% for V̇E, 1.05-3.79% for BF, 1.10%-17.5% for V̇O2 , 1.07%-18.3% for V̇CO2 , 0.62%-14.8% for RER, 5.52%-99.0% for Kcal from carbs, 5.13%-133% for Kcal from fats, and 0.59%-12.1% for total energy expenditure. Between-session variation ranged from 0.86%-22.4% for V̇O2 and 1.14%-20.2% for V̇CO2 , respectively. The error of respiratory gas variables, substrate, and energy use differed substantially between systems, with only a few systems demonstrating a consistent acceptable error. We extensively discuss the implications of our findings for clinicians, researchers and other CPET users.


Isolation of a snake venom phospholipase A2 (PLA2) inhibitor (AIPLAI) from leaves of Azadirachta indica (Neem): mechanism of PLA2 inhibition by AIPLAI in vitro condition.
A compound (AIPLAI (Azadirachta indica PLA(2) inhibitor)) purified from the methanolic leaf extract of A. indica (Neem) inhibits the cobra and Russell's viper venoms (RVVs) phospholipase A(2) enzymes in a dose-dependent manner. Inhibition of catalytic and tested pharmacological properties of cobra venom (Naja naja and Naja kaouthia) PLA(2) enzymes by AIPLAI is significantly higher (P<0.05) compared to the inhibition of PLA(2) enzymes of crude RVV (Daboia russelli) when tested under the same condition. Kinetic study reveals that in in vitro condition, AIPLAI inhibits the purified N. kaouthia PLA(2) enzymes in a non-competitive manner. The AIPLAI is quite stable at room temperature. The present study shows that AIPLAI holds good promise for the development of novel anti-snake venom drug in future.


Methylated genes in breast cancer: associations with clinical and histopathological features in a familial breast cancer cohort.
Hundreds of hypermethylated genes have been described in breast cancer, yet the nature and contribution of these genes in their methylated state to overall risk and prognosis is under-characterized in non-sporadic breast cancers. We therefore compared associations of DNA methylation with tumor stage, hormone/growth receptor status, and clinical outcomes in a familial breast cancer cohort. Because few previous methylation studies have considered the oncogenic or tumor suppressor properties of their gene sets, this functional status was included as part of our correlative analysis. We found methylation of oncogenes was associated with better prognostic indicators, whereas tumor suppressor gene methylation was associated with a more severe phenotype in women that were either HER2+ or lymph node positive at diagnosis, and/or tended to recur or develop distant metastases. For example, the methylation of the tumor suppressor gene APC was strongly associated with a specific subset of tumors that were both ER+ and HER2+, while methylation of the TWIST oncogene was associated with breast cancers that did not metastasize. This was a retrospective, hospital-based study of n = 99 archival breast tumors derived from women with a germline genetic BRCA1 or BRCA2 mutation and/or familial breast cancer history. DNA methylation was quantified from formalin fixed, paraffin embedded tumors using the established protocol of quantitative multiplex-methylation specific PCR (QM-MSP). Non-parametric statistics were used to analyze candidate gene methylation in association with clinical outcomes. We report several novel, positive associations between percent methylation of the APC, RASSF1A, TWIST, ERα, CDH1, and Cyclin D2 genes and key variables such as tumor stage, hormone and growth receptor status, and a history of recurrent or metastatic disease. Our data suggest the potential utility of parsing gene methylation by functional status and breast tumor subtype.


Sibshops: an evaluation of an interagency model.
The study evaluates the effectiveness of a sibling support programme (Sibshops) involving three disability agencies in Cork, Ireland. Qualitative and quantitative data were obtained using semi-structured interviews and the Piers-Harris Children's Self-Concept Scale with siblings, together with parent feedback. Results from the Piers-Harris showed that there was no significant increase in sibling self-esteem following attendance at the Sibshops. However, the interviews revealed that the majority of siblings enjoyed and benefited from the Sibshops. Parents reported satisfaction with the Sibshops and felt that their children had benefited. Reflections on the experience of working on an interagency basis are outlined. Recommendations are made regarding further research and development in the organization of Sibshops and in staffing and staff training.


The Liver in Children With Metabolic Syndrome.
Non-alcoholic fatty liver disease (NAFLD) is recognized as an emerging health risk in obese children and adolescents. NAFLD represents a wide spectrum of liver conditions, ranging from asymptomatic steatosis to steatohepatitis. The growing prevalence of fatty liver disease in children is associated with an increased risk of metabolic and cardiovascular complications. NAFLD is considered the hepatic manifestation of Metabolic Syndrome (MetS) and several lines of evidence have reported that children with NAFLD present one or more features of MetS. The pathogenetic mechanisms explaining the interrelationships between fatty liver disease and MetS are not clearly understood. Altough central obesity and insulin resistance seem to represent the core of the pathophysiology in both diseases, genetic susceptibility and enviromental triggers are emerging as crucial components promoting the development of NAFLD and MetS in children. In the present review we have identified and summarizied studies discussing current pathogenetic data of the association between NAFLD and MetS in children.


Robust discovery of gene regulatory networks from single-cell gene expression data by Causal Inference Using Composition of Transactions.
Gene regulatory networks (GRNs) drive organism structure and functions, so the discovery and characterization of GRNs is a major goal in biological research. However, accurate identification of causal regulatory connections and inference of GRNs using gene expression datasets, more recently from single-cell RNA-seq (scRNA-seq), has been challenging. Here we employ the innovative method of Causal Inference Using Composition of Transactions (CICT) to uncover GRNs from scRNA-seq data. The basis of CICT is that if all gene expressions were random, a non-random regulatory gene should induce its targets at levels different from the background random process, resulting in distinct patterns in the whole relevance network of gene-gene associations. CICT proposes novel network features derived from a relevance network, which enable any machine learning algorithm to predict causal regulatory edges and infer GRNs. We evaluated CICT using simulated and experimental scRNA-seq data in a well-established benchmarking pipeline and showed that CICT outperformed existing network inference methods representing diverse approaches with many-fold higher accuracy. Furthermore, we demonstrated that GRN inference with CICT was robust to different levels of sparsity in scRNA-seq data, the characteristics of data and ground truth, the choice of association measure and the complexity of the supervised machine learning algorithm. Our results suggest aiming at directly predicting causality to recover regulatory relationships in complex biological networks substantially improves accuracy in GRN inference.


Mutagenesis studies of the 14 Å internal cavity of histone deacetylase 1: insights toward the acetate-escape hypothesis and selective inhibitor design.
Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, as shown by the approval of two HDAC inhibitors for the treatment of cutaneous T-cell lymphoma. HDAC1 in particular has been linked to cell growth and cell cycle regulation and is therefore an attractive target for anticancer drugs. The HDAC1 active site contains a hydrophobic 11 Å active-site channel, with a 14 Å internal cavity at the bottom of the active site. Several computational and biochemical studies have proposed an acetate-escape hypothesis where the acetate byproduct of the deacetylation reaction escapes via the 14 Å internal cavity. Selective HDAC inhibitors that bind to the 14 Å cavity have also been created. To understand the influence of amino acids lining the HDAC1 14 Å cavity in acetate escape and inhibitor binding, we used mutagenesis coupled with acetate competition assays. The results indicate that amino acids lining the 14 Å cavity are critical for catalytic activity and acetate competition, confirming the role of the cavity in acetate escape. In addition, these mutagenesis studies will aid in HDAC1-inhibitor design that exploits the 14 Å cavity.


Premature termination of RNA polymerase II mediated transcription of a seed protein gene in Schizosaccharomyces pombe.
The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe. The 3'-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus. Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements. RT-PCR analyses detected transcripts up to the second SDE. This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe. The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions. Deletion of these cis-elements abolished 3'-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE. These results indicate that the bipartite sequence elements as signals for 3'-end processing in fission yeast act in tandem with other cis-acting elements. A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element. However, these sequences are not identical. Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611-949 nt. Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene.


A novel calcium-sensing domain in the BK channel.
The high-conductance Ca2+-activated K+ channel (mSlo) plays a vital role in regulating calcium entry in many cell types. mSlo channels behave like voltage-dependent channels, but their voltage range of activity is set by intracellular free calcium. The mSlo subunit has two parts: a "core" resembling a subunit from a voltage-dependent K+ channel, and an appended "tail" that plays a role in calcium sensing. Here we present evidence for a site on the tail that interacts with calcium. This site, the "calcium bowl," is a novel calcium-binding motif that includes a string of conserved aspartate residues. Mutations of the calcium bowl fall into two categories: 1) those that shift the position of the G-V relation a similar amount at all [Ca2+], and 2) those that shift the position of the G-V relation only at low [Ca2+]. None of these mutants alters the slope of the G-V curve. These mutant phenotypes are apparent in calcium ion, but not in cadmium ion, where mutant and wild type are indistinguishable. This suggests that the calcium bowl is sensitive to calcium ion, but insensitive to cadmium ion. The presence and independence of a second calcium-binding site is inferred because channels still respond to increasing levels of [Ca2+] or [Cd2+], even when the calcium bowl is mutationally deleted. Thus a low level of activation in the absence of divalent cations is identical in mutant and wild-type channels, possibly because of activation of this second Ca2+-binding site.


Biochemical and Spectroscopic Characterization of a Radical S-Adenosyl-L-methionine Enzyme Involved in the Formation of a Peptide Thioether Cross-Link.
Peptide-derived natural products are a class of metabolites that afford the producing organism a selective advantage over other organisms in their biological niche. While the polypeptide antibiotics produced by the nonribosomal polypeptide synthetases (NRPS) are the most widely recognized, the ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging group of natural products with diverse structures and biological functions. Both the NRPS derived peptides and the RiPPs undergo extensive post-translational modifications to produce structural diversity. Here we report the first characterization of the six cysteines in forty-five (SCIFF) [Haft, D. H. and Basu M. K. (2011) J. Bacteriol. 193, 2745-2755] peptide maturase Tte1186, which is a member of the radical S-adenosyl-l-methionine (SAM) superfamily. Tte1186 catalyzes the formation of a thioether cross-link in the peptide Tte1186a encoded by an orf located upstream of the maturase, under reducing conditions in the presence of SAM. Tte1186 contains three [4Fe-4S] clusters that are indispensable for thioether cross-link formation; however, only one cluster catalyzes the reductive cleavage of SAM. Mechanistic imperatives for the reaction catalyzed by the thioether forming radical SAM maturases will be discussed.


Looking At the Past and Heading to the Future: Meeting Summary of the 6th European Workshop on Plant Chromatin 2019 in Cologne, Germany.
In June 2019, more than a hundred plant researchers met in Cologne, Germany, for the 6th European Workshop on Plant Chromatin (EWPC). This conference brought together a highly dynamic community of researchers with the common aim to understand how chromatin organization controls gene expression, development, and plant responses to the environment. New evidence showing how epigenetic states are set, perpetuated, and inherited were presented, and novel data related to the three-dimensional organization of chromatin within the nucleus were discussed. At the level of the nucleosome, its composition by different histone variants and their specialized histone deposition complexes were addressed as well as the mechanisms involved in histone post-translational modifications and their role in gene expression. The keynote lecture on plant DNA methylation by Julie Law (SALK Institute) and the tribute session to Lars Hennig, honoring the memory of one of the founders of the EWPC who contributed to promote the plant chromatin and epigenetic field in Europe, added a very special note to this gathering. In this perspective article we summarize some of the most outstanding data and advances on plant chromatin research presented at this workshop.


Reported reasons for not using a mosquito net when one is available: a review of the published literature.
A review of the barriers to mosquito net use in malaria-endemic countries has yet to be presented in the published literature despite considerable research interest in this area. This paper partly addresses this gap by reviewing one component of the evidence base; namely, published research pertaining to self-reported reasons for not using a mosquito net among net 'owning' individuals. It was anticipated that the review findings would potentially inform an intervention or range of interventions best suited to promoting greater net use amongst this group. Studies were sought via a search of the Medline database. The key inclusion criteria were: that study participants could be identified as owning a mosquito net or having a mosquito net available for use; that these participants on one or more occasions were identified or self-reported as not using the mosquito net; and that reasons for not using the mosquito net were reported. Studies meeting these criteria were included irrespective of mosquito net type. A total of 22 studies met the inclusion criteria. Discomfort, primarily due to heat, and perceived (low) mosquito density were the most widely identified reason for non-use. Social factors, such as sleeping elsewhere, or not sleeping at all, were also reported across studies as were technical factors related to mosquito net use (i.e. not being able to hang a mosquito net or finding it inconvenient to hang) and the temporary unavailability of a normally available mosquito net (primarily due to someone else using it). However, confidence in the reported findings was substantially undermined by a range of methodological limitations and a dearth of dedicated research investigation. The findings of this review should be considered highly tentative until such time as greater quantities of dedicated, well-designed and reported studies are available in the published literature. The current evidence-base is not sufficient in scope or quality to reliably inform mosquito net promoting interventions or campaigns targeted at individuals who own, but do not (reliably) use, mosquito nets.


A neural field model of the somatosensory cortex: formation, maintenance and reorganization of ordered topographic maps.
We investigate the formation and maintenance of ordered topographic maps in the primary somatosensory cortex as well as the reorganization of representations after sensory deprivation or cortical lesion. We consider both the critical period (postnatal) where representations are shaped and the post-critical period where representations are maintained and possibly reorganized. We hypothesize that feed-forward thalamocortical connections are an adequate site of plasticity while cortico-cortical connections are believed to drive a competitive mechanism that is critical for learning. We model a small skin patch located on the distal phalangeal surface of a digit as a set of 256 Merkel ending complexes (MEC) that feed a computational model of the primary somatosensory cortex (area 3b). This model is a two-dimensional neural field where spatially localized solutions (a.k.a. bumps) drive cortical plasticity through a Hebbian-like learning rule. Simulations explain the initial formation of ordered representations following repetitive and random stimulations of the skin patch. Skin lesions as well as cortical lesions are also studied and results confirm the possibility to reorganize representations using the same learning rule and depending on the type of the lesion. For severe lesions, the model suggests that cortico-cortical connections may play an important role in complete recovery.


Effects of 25 mg oxazepam on emotional mimicry and empathy for pain: a randomized controlled experiment.
Emotional mimicry and empathy are mechanisms underlying social interaction. Benzodiazepines have been proposed to inhibit empathy and promote antisocial behaviour. First, we aimed to investigate the effects of oxazepam on emotional mimicry and empathy for pain, and second, we aimed to investigate the association of personality traits to emotional mimicry and empathy. Participants (n=76) were randomized to 25 mg oxazepam or placebo. Emotional mimicry was examined using video clips with emotional expressions. Empathy was investigated by pain stimulating the participant and a confederate. We recorded self-rated experience, activity in major zygomatic and superciliary corrugator muscles, skin conductance, and heart rate. In the mimicry experiment, oxazepam inhibited corrugator activity. In the empathy experiment, oxazepam caused increased self-rated unpleasantness and skin conductance. However, oxazepam specifically inhibited neither emotional mimicry nor empathy for pain. Responses in both experiments were associated with self-rated empathic, psychopathic and alexithymic traits. The present results do not support a specific effect of 25 mg oxazepam on emotional mimicry or empathy.


Aging and DNA methylation in colorectal mucosa and cancer.
DNA methylation of promoter-associated CpG islands may function as an alternate mechanism of silencing tumor suppressor genes in multiple neoplasias including colorectal cancer. De novo methylation of genes appears to be an early and frequent event in most neoplasias. For the ER and IGF2 genes, we have previously shown that methylation actually begins in the normal colon mucosa as an age-related event and progresses to hypermethylation in cancer. In this study, we have determined the frequency of age-related methylation in normal colonic mucosa among the genes hypermethylated in colorectal cancer. We studied six genes, including N33, MYOD, p16, HIC-1, THBS1, and CALCA. The N33 gene showed partial methylation in normal colon mucosa, which was age-related (r = 0.7; P = 0.003 using regression analysis). Adenomas and cancers showed further hypermethylation at this locus. Similarly, the MYOD gene showed age-related methylation in normal colon mucosa (r = 0.7; P < 0.00001 using regression analysis) and hypermethylation in cancers. Age-related methylation seems to be gene specific, because p16, THBS1, HIC-1, and CALCA were not affected. Furthermore, this process may also be modulated by tissue-specific factors. Our study suggests that aging is a major contributing factor to hypermethylation in cancer.


Inside athletes' minds: preliminary results from a pilot study on mental representation of doping and potential implications for anti-doping.
Despite the growing body of literature and putative links between the use of ergogenic nutritional supplements, doping and illicit drugs, it remains unclear whether, in athletes' minds, doping aligns with illicit behaviour or with functional use of chemical or natural preparations. To date, no attempt has been made to quantitatively explore athletes' mental representation of doping in relation to illegality and functionality. A convenience sample of student athletes from a large South-Eastern Australian university responded to an on-line survey. Competitive athletes (n = 46) were grouped based on self-reported use as follows: i) none used (30%), ii) supplement only (22%), iii) illicit only (26%) and iv) both supplements and illicit drug use (22%). Whereas no athlete reported doping, data provided on projected supplement-, doping- and drug use by the four user groups allowed evaluation of doping-related cognition in the context of self-reported supplement- and illicit drug taking behaviour; and comparison between these substances. A significantly higher prevalence estimation was found for illicit drug use and a trend towards a biased social projection emerged for supplement use. Doping estimates by user groups showed mixed results, suggesting that doping had more in common with the ergogenic nutritional supplement domain than the illicit drug domain. Assessing the behavioural domain to which doping belongs to in athletes' mind would greatly advance doping behaviour research toward prevention and intervention. Further investigation refining the peculiarity of the mental representation of doping with a larger study sample, controlling for knowledge of doping and other factors, is warranted.


A2 diagnostic criterion for combat-related posttraumatic stress disorder.
Individuals trained to respond to a potentially traumatic event may not experience the posttraumatic stress disorder (PTSD) A2 diagnostic criterion of fear, helplessness, or horror and yet may still report significant PTSD symptoms. The present study included interviews with 202 soldiers returning from a year in Iraq. Although reporting an A2 response was associated with higher PTSD Checklist scores, there were no significant differences in the percentage of subjects who met cutoff criteria for PTSD. The most common alternative A2 responses were related to military training and anger. The A2 criterion for PTSD should be expanded so as not to underestimate the number of individuals trained for high-risk occupations who might benefit from treatment.


Early leucine programming on protein utilization and mTOR signaling by DNA methylation in zebrafish (Danio rerio).
Early nutritional programming affects a series of metabolism, growth and development in mammals. Fish also exhibit the developmental plasticity by early nutritional programming. However, little is known about the effect of early amino acid programming on growth and metabolism. In the present study, zebrafish (Danio rerio) was used as the experimental animal to study whether early leucine stimulation can programmatically affect the mechanistic target of rapamycin (mTOR) signaling pathway, growth and metabolism in the later life, and to undercover the mechanism of epigenetic regulation. Zebrafish larvas at 3 days post hatching (dph) were raised with 1.0% leucine from 3 to 13 dph during the critical developmental stage, then back to normal water for 70 days (83 dph). The growth performance and crude protein content of zebrafish in the early leucine programming group were increased, and consistent with the activation of the mTOR signaling pathway and the high expression of genes involved in the metabolism of amino acid and glycolipid. Furthermore, we compared the DNA methylation profiles between the control and leucine-stimulated zebrafish, and found that the methylation levels of CG-differentially methylated regions (DMGs) and CHH-DMGs of genes involved in mTOR signaling pathway were different between the two groups. With quantitative PCR analysis, the decreased methylation levels of CG type of Growth factor receptor-bound protein 10 (Grb10), eukaryotic translation initiation factor 4E (eIF4E) and mTOR genes of mTOR signaling pathway in the leucine programming group, might contribute to the enhanced gene expression. The early leucine programming could improve the protein synthesis and growth, which might be attributed to the methylation of genes in mTOR pathway and the expression of genes involved in protein synthesis and glycolipid metabolism in zebrafish. These results could be beneficial for better understanding of the epigenetic regulatory mechanism of early nutritional programming.


Overexpression of a novel E3 ubiquitin ligase gene from Coptis chinensis Franch enhances drought tolerance in transgenic tobacco.
Drought stress has a significant effect on the growth, physiology and biochemistry of medicinal plants. SDIR1 (Salt- and Drought-Induced Ring Finger1), a C3H2C3-type RING-finger E3 ubiquitin ligase gene plays an important role in the stress response of various plants. However, the role of this gene is not clear in Coptis chinensis. In this study, the CcSDIR1 gene was cloned from C. chinensis using RACE and RT-PCR. Sequence analysis revealed that CcSDIR1 had an open reading frame of 840 bp that encodes 279 amino acids with a theoretical molecular weight about 31 kDa and pI value of 5.65 and shared conserved domains with other plants. On comparison with the wild-type plants, overexpression of CcSDIR1 in transgenic tobaccos increased drought tolerance and showed better growth performance. However, lower malondialdehyde contents and high antioxidant enzyme activities were observed in transgenic tobacco plants compared to wild-type plants. In addition, Evans blue staining showed high cell viability of transgenic lines under drought stress. These results suggest that CcSDIR1 regulates various responses to drought stress by increasing antioxidant enzyme activities and reducing oxidative damage. From the study results, the CcSDIR1 gene will be very useful for drought stress research in plants.


Diagnosis of gastrointestinal tract lesions by endoscopic ultrasound-guided fine-needle aspiration biopsy.
Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) allows detailed imaging of both intramural and extramural structures of the gastrointestinal (GI) tract and also allows tissue samples to be obtained from masses and lesions in the GI tract. The objective of the current study was to determine the diagnostic utility of EUS-FNA in evaluating intramural and extramural GI tract lesions. The authors evaluated all EUS-FNA specimens of GI tract lesions obtained over a 30-month period (from August 2000 to February 2003). Samples of pancreatic and intrabdominal/mediastinal lymph nodes were excluded from the study. A single endosonographer performed all procedures. An attending cytopathologist also was present on site to assess specimen adequacy. Cytologic diagnoses were analyzed for correlations with final diagnoses, which were based on histologic examination of biopsied/resected pathology materials and/or clinical follow-up findings. Sixty-two EUS-FNA specimens of intramural and extramural GI tract lesions were obtained from a total of 60 patients. The mean patient age was 58.8 years (standard deviation, 15.3 years). Thirty-six patients (60%) were male, and 24 (40%) were female. Twenty-eight patients had surgical pathologic evaluation of the corresponding lesions. The remaining 32 patients were followed clinically for a mean duration of 9.5 months (standard deviation, 7.7 months). The anatomic sites of the lesions were as follows: esophagus in 23 patients (37%), stomach in 13 patients (21%), duodenum in 15 patients (24%), and rectum/sigmoid in 11 patients (18%). It is noteworthy that 29 patients (43%) previously had experienced unsuccessful attempts at tissue diagnosis by endoscopic forceps biopsy. Of the 62 EUS-FNA specimens, 43, 4, and 15 were reported as being positive for a neoplasm, suspicious, and benign, respectively. Neoplastic lesions included carcinoma (n = 24), gastrointestinal stromal tumor (GIST; n = 18), neuroendocrine neoplasm (n = 2), and lymphoma (n = 1). There were two cases of endometriosis, three foregut duplication cysts, and one case of diverticulosis. There were two lesions that yielded false-negative findings (one gastric lymphoma and one GIST) secondary to sampling or interpretive error. There also were three cases that yielded false-positive findings (one case of endometriosis, one case of duodenal diverticula with smooth muscle hyperplasia, and one case of normal pancreas, which presented as a periduodenal mass). The sensitivity, specificity, and diagnostic accuracy of EUS-FNA in diagnosing GI tract neoplastic lesions were 89%, 88%, and 89%, respectively. EUS-FNA provides accurate tissue diagnosis in a wide variety of extraintestinal mass lesions and intramural GI tumors, particularly in patients for whom previous endoscopic forceps biopsy was unsuccessful in establishing a diagnosis.


Modelling cyanobacteria: from metabolism to integrative models of phototrophic growth.
Cyanobacteria are phototrophic microorganisms of global importance and have recently attracted increasing attention due to their capability to convert sunlight and atmospheric CO(2) directly into organic compounds, including carbon-based biofuels. The utilization of cyanobacteria as a biological chassis to generate third-generation biofuels would greatly benefit from an increased understanding of cyanobacterial metabolism and its interplay with other cellular processes. In this respect, metabolic modelling has been proposed as a way to overcome the traditional trial and error methodology that is often employed to introduce novel pathways. In particular, flux balance analysis and related methods have proved to be powerful tools to investigate the organization of large-scale metabolic networks-with the prospect of predicting modifications that are likely to increase the yield of a desired product and thereby to streamline the experimental progress and avoid futile avenues. This contribution seeks to describe the utilization of metabolic modelling as a research tool to understand the metabolism and phototrophic growth of cyanobacteria. The focus of the contribution is on a mathematical description of the metabolic network of Synechocystis sp. PCC 6803 and its analysis using constraint-based methods. A particular challenge is to integrate the description of the metabolic network with other cellular processes, such as the circadian clock, the photosynthetic light reactions, carbon concentration mechanism, and transcriptional regulation-aiming at a predictive model of a cyanobacterium in silico.


Ultrastable substrates for surface-enhanced Raman spectroscopy: Al2O3 overlayers fabricated by atomic layer deposition yield improved anthrax biomarker detection.
A new method to stabilize and functionalize surfaces for surface-enhanced Raman spectroscopy (SERS) is demonstrated. Atomic layer deposition (ALD) is used to deposit a sub-1-nm alumina layer on silver film-over-nanosphere (AgFON) substrates. The resulting overlayer maintains and stabilizes the SERS activity of the underlying silver while presenting the surface chemistry of the alumina overlayer, a commonly used polar adsorbent in chromatographic separations. The relative affinity of analytes for alumina-modified AgFON substrates can be determined by their polarity. On the basis of SERS measurements, dipicolinic acid displays the strongest binding to the ALD alumina-modified AgFON among a set of pyridine derivatives with varying polarity. This strong affinity for carboxylate groups makes the SERS substrate an ideal candidate for bacillus spores detection using the dipicolinate biomarker. The SERS signal from extracted dipicolinate was measured over the spore concentration range 10(-14)-10(-12) M to determine the saturation binding capacity of the alumina-modified AgFON surface. The adsorption constant was determined to be Kspore = 9.0 x 10(13) M(-1). A 10-s data collection time is capable of achieving a limit of detection of approximately 1.4 x 10(3) spores. The shelf life of prefabricated substrates is at least 9 months prior to use. In comparison to the bare AgFON substrates, the ALD-modified AgFON substrates demonstrate twice the sensitivity with 6 times shorter data acquisition time and 7 times longer temporal stability. ALD expands the palette of available chemical methods to functionalize SERS substrates, which will enable improved and diverse chemical control over the nature of analyte-surface binding for biomedical, homeland security, and environmental applications.


Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane.
A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.


AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans.
Dynein complexes are large, multi-unit assemblies involved in many biological processes including male fertility via their critical roles in protein transport and axoneme motility. Previously we identified a pathogenic variant in the dynein gene AXDND1 in an infertile man. Subsequently we identified an additional four potentially compound heterozygous variants of unknown significance in AXDND1 in two additional infertile men. We thus tested the role of AXDND1 in mammalian male fertility by generating a knockout mouse model. Axdnd1-/- males were sterile at all ages but could undergo one round of histologically complete spermatogenesis. Subsequently, a progressive imbalance of spermatogonial commitment to spermatogenesis over self-renewal occurred, ultimately leading to catastrophic germ cell loss, loss of blood-testis barrier patency and immune cell infiltration. Sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively, our data highlight the essential roles of AXDND1 as a regulator of spermatogonial commitment to spermatogenesis and during the processes of spermiogenesis where it is essential for sperm tail development, release and motility.


Dissimilar interaction of factor VIII with endothelial cells and lipid vesicles during factor X activation.
A localized and regulated cascade of proteolytic events is a prerequisite for normal haemostasis. The activation of factor X by activated factor IX (factor IXa) in the presence of activated factor VIII (factor VIIIa) is essential for the formation of a fibrin clot at sites of vascular injury. We observed sustained activation of factor X on the surface of vascular endothelial cells, whereas, in agreement with others, on synthetic negatively charged phospholipid vesicles and activated blood platelets factor X activation is transient and starts to decline a few minutes after the onset of the reaction. We examined the mechanism responsible for these differences in factor X activation. Procoagulant membrane and solution were analysed separately for the occurrence of factor VIII and its activation fragments. On negatively charged phospholipid vesicles, on dissociation of factor VIIIa, the 67 kDa light-chain fragment remains associated with the lipid membrane. As a result, factor VIII-binding sites remain occupied, and dampening of factor X activation occurs. In contrast, on monolayers of endothelial cells, no residual factor VIIIa fragments associated with the cell membrane were observed. During endothelial-cell-mediated activation of factor X, accumulation of factor VIIIa fragments was observed in the solution phase only. This finding suggests that, on endothelial cells, factor VIII-binding sites remain accessible for further factor VIII binding, guaranteeing sustained activation of factor X. These data demonstrate that the nature of the procoagulant membrane contributes to the regulation of the cofactor activity of factor VIII and thereby affects the progress of factor X activation.


Mechanisms of T cell peptide epitope-dependent late asthmatic reactions.
Short peptide sequences corresponding to T cell epitopes have been identified in the major cat allergen Fel d 1. In order to directly activate allergen-specific T cells in cat-allergic asthmatic individuals, peptides were administered by intradermal injection. Subsequently, a proportion of subjects experienced a delayed reduction of airway calibre manifested as a decrease in FEV(1). Changes in lung function occurred approximately 3 h after peptide injection, peaked at 6 h and resembled an isolated late asthmatic reaction (LAR). Using molecular tissue typing techniques, it was determined that many of the individuals experiencing isolated LAR expressed particular HLA-DR molecules. These molecules were shown in subsequent experiments to bind individual peptides within the preparation and thus to activate T cells in a major histocompatibility complex (MHC)-restricted fashion. The precise mechanisms whereby MHC-restricted activation of allergen-specific T cells gives rise to bronchoconstriction are currently under investigation.


Blocking early GABA depolarization with bumetanide results in permanent alterations in cortical circuits and sensorimotor gating deficits.
A high incidence of seizures occurs during the neonatal period when immature networks are hyperexcitable and susceptible to hypersyncrhonous activity. During development, γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in adults, typically excites neurons due to high expression of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). NKCC1 facilitates seizures because it renders GABA activity excitatory through intracellular Cl(-) accumulation, while blocking NKCC1 with bumetanide suppresses seizures. Bumetanide is currently being tested in clinical trials for treatment of neonatal seizures. By blocking NKCC1 with bumetanide during cortical development, we found a critical period for the development of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate synapses. Disruption of GABA signaling during this window resulted in permanent decreases in excitatory synaptic transmission and sensorimotor gating deficits, a common feature in schizophrenia. Our study identifies an essential role for GABA-mediated depolarization in regulating the balance between cortical excitation and inhibition during a critical period and suggests a cautionary approach for using bumetanide in treating neonatal seizures.


Lamellar Macular Hole: Two Distinct Clinical Entities?
To investigate whether lamellar macular holes can be divided into different subgroups. Retrospective observational case series. In this institutional study, clinical charts and spectral-domain optical coherence tomography (OCT) images of 102 eyes of 90 consecutive patients diagnosed with lamellar macular hole were reviewed. In OCT imaging, the presence of lamellar macular hole was defined according to the following findings: presence of irregular foveal contour, separation of the layers of the neurosensory retina, and the absence of full-thickness macular defect. Mean outcome was the morphologic and functional characterization of different subtypes of macular hole. Two different subtypes of lamellar macular hole were identified: tractional and degenerative. The first type, tractional, was diagnosed in 43 eyes, and was characterized by the schitic separation of neurosensory retina between outer plexiform and outer nuclear layers. It often presented with an intact ellipsoid layer and was associated with tractional epiretinal membranes and/or vitreomacular traction. The second type, degenerative, was diagnosed in 48 eyes, and its distinctive traits included the presence of intraretinal cavitation that could affect all retinal layers. It was often associated with nontractional epiretinal proliferation and a retinal "bump." Moreover, it often presented with early ellipsoidal zone defect and its pathogenesis, although chronic and progressive, remains poorly understood. Eleven eyes shared common features with both tractional and degenerative lamellar macular holes and were classified as mixed lesions. Degenerative and tractional lamellar macular holes may be 2 distinct clinical entities. A revision of the current concept of lamellar macular holes is needed.