Endocrine disruptors as a threat to neurological function.
Endocrine disruption is a concept and principle whose origins can be traced to the beginnings of the environmental movement in the 1960s. It began with puzzlement about and the flaring of research on the decline of wildlife, particularly avian species. The proposed causes accented pesticides, especially persistent organochlorines such as DDT. Its scope gradually widened beyond pesticides, and, as endocrine disruption offered an explanation for the wildlife phenomena, it seemed to explain, as well, changes in fertility and disorders of male reproduction such as testicular cancer. Once disturbed gonadal hormone function became the most likely explanation, it provoked other questions. The most challenging arose because of how critical gonadal hormones are to brain function, especially as determinants of brain sexual differentiation. Pursuit of such connections has generated a robust literature embracing a broad swath of chemical classes. How endocrine disrupting chemicals influence the adult and aging brain is a question, so far mostly ignored because of the emphasis on early development, that warrants vigorous investigation. Gonadal hormones are crucial to optimal brain function during maturity and even senescence. They are pivotal to the processes of neurogenesis. They exert protective actions against neurodegenerative disorders such as dementia and support smoothly functioning cognitive activities. The limited research conducted so far on endocrine disruptors, aging, and neurogenesis argues that they should be overlooked no longer.


In silico design of additively manufacturable composite synthetic vascular conduits and grafts with tuneable compliance.
Benchtop testing of endovascular medical devices under accurately simulated physiological conditions is a critical part of device evaluation prior to clinical assessment. Currently, glass, acrylic and silicone vascular models are predominantly used as anatomical simulator test beds for in vitro testing. However, most current models lack the ability to mimic the non-linear radial compliance of native vessels and are typically limited to being compliance-matched at a single mean pressure comparison point or not at all. Hence, a degree of caution needs to be shown when analysing results from such models under simulated physiological or pathophysiological conditions. Similarly, the clinical translation of proposed biomimetic compliance-matched vascular grafts has undoubtedly been curtailed due to performance and material limitations. Here, we propose a new design for synthetic vessels where compliance can be precisely modulated across a wide physiological pressure range by customising design parameters. Building on previously demonstrated methods of 3D printing composite compliant cylindrical structures, we demonstrate proof of principle in creating composite vascular constructs designed via a finite element model. Our constructs are 3D printable and consist of a soft silicone matrix with embedded polyurethane fibres. The fibre layer consists of circumferential sinusoidal waves with an amplitude that can be altered to result in tuneable internal radial compliances of 5.2-15.9%/mmHg × 10-2 at a mean pressure of 100 mmHg. Importantly, the design presented here allows preservation of the non-linear exponentially decaying compliance curve of native arteries and veins with an increasing mean pressure. This model offers a design toolbox for 3D printable vascular models that offer biomimetic compliance. The robust nature of this model will lead to rapidly accelerating the design process for biomimetic vascular anatomical simulators, lumped parameter model flow loops, endovascular device benchtop testbeds, and compliance-matched synthetic grafts.


VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts.
Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.


Oxidation of nitric oxide by oxomanganese-salen complexes: a new mechanism for cellular protection by superoxide dismutase/catalase mimetics.
Manganese-salen complexes (Mn-Salen), including EUK-8 [manganese N,N'-bis(salicylidene)ethylenediamine chloride] and EUK-134 [manganese 3-methoxy N,N'-bis(salicylidene)ethylenediamine chloride], have been reported to possess combined superoxide dismutase (SOD) and catalase mimetic functions. Because of this SOD/catalase mimicry, EUK-8 and EUK-134 have been investigated as possible therapeutic agents in neurological disorders resulting from oxidative stress, including Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. These actions have been explained by the ability of the Mn-Salen to remove deleterious superoxide (O(2)(-)) and H(2)O(2). However, in addition to oxidative stress, cells in models for neurodegenerative diseases may also be subjected to damage from reactive nitrogen oxides (nitrosative stress), resulting from elevated levels of NO and sister compounds, including peroxynitrite (ONOO(-)). We have been examining the interaction of EUK-8 and EUK-134 with NO and ONOO(-). We find that in the presence of a per-species (H(2)O(2), ONOO(-), peracetate and persulphate), the Mn-Salen complexes are oxidized to the corresponding oxo-species (oxoMn-Salen). OxoMn-Salens are potent oxidants, and we demonstrate that they can rapidly oxidize NO to NO(2) and also oxidize nitrite (NO(2)(-) to nitrate (NO(2)(-)). Thus these Mn-Salens have the potential to ameliorate cellular damage caused by both oxidative and nitrosative stresses, by the catalytic breakdown of O(2)(-), H(2)O(2), ONOO(-) and NO to benign species: O(2), H(2)O, NO(2)(-) and NO(3)(-).


Visually perceived eye level with reversible pitch stimuli: implications for the great circle and implicit surface models.
Visually perceived eye level (VPEL) and perceived pitch were measured while subjects viewed two sets of stimuli that were either upright or pitched top-toward or top-away from them. The first set of stimuli, a pair of vertical lines viewed at various angles of pitch, caused systematic changes in perceived pitch and upward and downward VPEL shifts for the top-toward and top-away pitches, respectively. Neither the perceived pitch nor the VPEL measures with these stimuli differed between monocular and binocular viewing. The second set of stimuli was constructed so that, when viewed at the appropriate pitch angle, the projected orientations of the lines in the retinal image of each stimulus were similar to those generated by a pair of vertical lines pitched by a lesser amount in the opposite direction. When viewed monocularly, these stimuli appeared pitched in the direction opposite their physical pitch, yet produced VPEL shifts consistent with the direction of their physical pitch. These results clearly demonstrate a dissociation between perceived pitch and VPEL. The same stimuli, when viewed binocularly, appeared pitched in the direction of their physical pitch and caused VPEL shifts indistinguishable from those obtained monocularly. The retinal image orientations of these stimuli, however, corresponded to those of vertical line stimuli pitched in the opposite direction. This finding is therefore consistent with the hypothesis that VPEL and perceived pitch are processed independently, but inconsistent with the specific version of this hypothesis which states that differences in VPEL are determined solely on the basis of the orientation of lines in the retinal image.


Nonpharmacologic treatments of bulimia nervosa.
The principal psychosocial approach to the treatment of bulimia nervosa has been cognitive-behavioral therapy, the effectiveness of which has been extensively documented in controlled trials, with full recovery (cessation of binge eating and purging) in some 50% to 60% of patients. More recently, interpersonal therapy, first introduced for the treatment of depression, has been shown in preliminary studies to be as effective as cognitive-behavioral therapy, particularly at follow-up. The evidence for effectiveness of psychosocial therapies in bulimia nervosa is first reviewed, and consideration is then given to what is known concerning the combination of psychosocial and psychopharmacologic treatment approaches and to the overall implications for the treatment of bulimia nervosa.


In vitro fitting accuracy of copy-milled alumina cores and all-ceramic crowns.
The purpose of this study was to evaluate the marginal and internal fit of alumina cores and all-ceramic crowns in a new preparation design for abutment teeth devised for copy milling. Using conventional dies and dies from the new type of preparation design, 20 alumina cores were produced by copy milling, 10 cores in each die type. Also using the new type of die, another 10 final crowns were fabricated. Each of the 30 specimens was returned to its original die and embedded in epoxy resin. Each specimen was then sectioned labiolingually and measured for gaps at the margin, in the central area, and at the incisal edge. The alumina cores fabricated using the new type of die had mean gaps of 30 to 40 microns at the margins of the labial and lingual sides, which was significantly smaller than the gaps (67 to 130 microns) found in the alumina cores produced with the conventional type of die. The final crowns had mean gaps of 44 to 63 microns at the margins and on the inner surfaces. There was no significant difference in the gaps between the alumina cores produced using the new type of die and those found in the final crowns. The new type of die was effective in allowing the fabrication of all-ceramic crowns that provide excellent marginal and internal fit by copy milling.


Characterization of a photoswitching chelator with light-modulated geometric, electronic, and metal-binding properties.
Photoswitching molecules are utilized for a variety of applications where the rapid manipulation of the molecules' chemical properties and spatial orientations allows for new spatiotemporal control over molecular-scale interactions and processes. Here, we present a hydrazone-containing transition metal chelator, HAPI ((E)-N'-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide), that displays dual-wavelength photoswitching behavior. Several of its metal complexes, however, are inert to photoreaction and thereby add another layer of control over the photoswitch system. The light-induced twist in HAPI structure is accompanied by a dramatic change in electronic properties as well as chelator strength. This work introduces HAPI as the prototype for a class of molecules with properties that may be optimized for a variety of experimental applications that take advantage of phototriggered molecular changes.


Properties of phosphoenolpyruvate mutase, the first enzyme in the aminoethylphosphonate biosynthetic pathway in Trypanosoma cruzi.
Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds. The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi. Using partial sequence information from the T. cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis. Recombinant expression in Escherichia coli confirms that it encodes a functional PEP mutase with a Km apparent of 8 microM for phosphonopyruvate and a kcat of 12 s-1. The native enzyme is a homotetramer with an absolute requirement for divalent metal ions and displays negative cooperativity for Mg2+ (S0.5 0.4 microM; n = 0.46). Immunofluorescence and sub-cellular fractionation indicates that PEP mutase has a dual localization in the cell. Further evidence to support this was obtained by Western analysis of a partial sub-cellular fractionation of T. cruzi cells. Southern and Western analysis suggests that PEP mutase is unique to T. cruzi and is not present in the other medically important parasites, Trypanosoma brucei and Leishmania spp.


An abattoir study on hepatic tumors of sheep.
The aim of the present study was to assess the incidence and age distribution of hepatic tumors of sheep as well as study of their histopathologic characteristics. For this purpose, 3000 slaughtered sheep, consisting of 284 rams with approximate age of 7 to 20 months and 2716 ewes with approximate age of 3 to 7 years, were inspected during a routine postmortem examination. This study was conducted at Tabriz abattoir in the East Azerbaijan province of Iran, in a course of 8 months from March to November 2006. In this survey, two livers were encountered tumoral. Representative sections of the tumors were stained with hematoxylin and eosin. Gross and microscopic features of these tumors supported diagnosis of hepatocellular carcinoma and cholangiocellular carcinoma for a seven-years-old crossbreed ewe and a five-years-old native breed ewe respectively. In this survey, 0.074% of slaughtered ewes had hepatic neoplasia. That is, the prevalence of hepatocellular carcinoma and cholangiocellular carcinoma in aged ewes was equally 0.037% in this study.


Histological and histomorphometric analyses of narrow implants, crestal and subcrestally placed in severe alveolar atrophy: a study in foxhound dogs.
Narrow-diameter implants provide an alternative to the horizontal augmentation techniques situations severe bone atrophy. Lack of bone width and interdental space has been regarded as an encumbrance in the case selection for prosthetic rehabilitation using dental implants. The aim of the study was to evaluate bone-to-implant contact and marginal bone loss of two different narrow implants in resorbed ridges at different crestal positions. 48 Bredent Narrow(®) implants (24 MiniSky(®) and NarowSky(®) ) were placed at crestal and subcrestal levels in healing bone of atrophic alveolar ridge of 6 American foxhounds. Histological and histomorphometric analyses of osseointegration were carried out at 4 and 8 weeks. Modeling in the marginal defect region was accompanied by marked decreases in the dimensions of both the buccal and the more lingual bone walls. Relative to BIC, significant differences were found in favor of subcrestal group, for both Narrow Sky and MiniSky. Linear measurements showed to be slightly high in the crestal group. With significant differences for PM-IS (peri-implant mucosa to implant shoulder) and IS-BC (implant shoulder to buccal bone crest). Within the limitations of animal study, it can be stated that the NarrowSky(test group) crestal and subcrestal implants showed less crestal bone resorption, higher ISQ values and most BIC at 4 and 8 weeks of evaluation compared with MiniSky implants (control group). The design of the implants plays an important role in peri-implant mucosa and crestal bone maintenance at 8-week follow-up period.


Will it be feasible to insert endoprostheses under interventional MRI?
Recent advances in magnetic resonance imaging (MRI) technology may provide a safer and more sensitive monitoring modality than X-ray imaging for endovascular surgical procedures. The purpose of this study was to investigate the feasibility of using MRI to monitor the insertion of endoprostheses. The endoprostheses we studied were composed of a nitinol stent encased in a polyester sheath. These were characterized with four different MRI techniques: the fast spin-echo; spin-echo; gradient-recalled echo; and the spoiled gradient-recalled echo. The deployment of the endoprosthesis into an artery was simulated in an in vitro model and viewed using a fast spin-echo MRI technique. Image artifacts produced by the nitinol framework in these endoprostheses were minimal when fast spin-echo or spin-echo imaging techniques were used, improving the visibility of the device. In in vitro tests, the catheters and endoprostheses were visualized by MRI with sufficient clarity to guide the placement of a device in the model artery. Insertion of this type of endoprosthesis under interventional MRI guidance is feasible. The convenience and improved safety provided by interventional MR systems and "real-time" imaging capabilities are expected to make this technology an attractive alternative to X-ray imaging techniques.


A URA3 null mutant of Candida albicans (CAI-4) causes oro-oesophageal and gastric candidiasis and is lethal for gnotobiotic, transgenic mice (Tgepsilon26) that are deficient in both natural killer and T cells.
Current data suggest that functional URA3 genes are necessary for the full pathogenesis of Candida albicans. Herein it is shown that a putatively avirulent URA3/URA3 null mutant of C. albicans (CAI-4) can colonize the murine alimentary tract, invade oro-oesophageal and gastric tissues with yeasts and hyphae, evoke a granulocyte-dominated inflammatory response, and kill transgenic mice that are deficient for both natural killer cells and T cells. Because C. albicans-colonized (gnotobiotic) mice lack a viable prokaryotic microbiota, this study also demonstrates that the gut microbiome is not required to supply the mutant's nutritional needs. The gnotobiotic murine model described herein can be used to assess the capacity of C. albicans mutants to colonize and infect cutaneous, mucosal and systemic tissues and kill the susceptible host via a clinically common, natural route of infection; namely the alimentary tract.


The association between biocide tolerance and the presence or absence of qac genes among hospital-acquired and community-acquired MRSA isolates.
The MBCs of three commonly used hospital biocides [containing quaternary ammonium compounds (QACs), chlorhexidine gluconate and triclosan] were determined for clinical isolates of Staphylococcus aureus, which were also screened for genes encoding Qac efflux pumps. MBCs were determined by broth microdilution for 94 clinical isolates of S. aureus, including 38 hospital-acquired methicillin-resistant S. aureus (HA-MRSA), 25 community-associated methicillin-resistant S. aureus (CA-MRSA), 25 methicillin-susceptible S. aureus (MSSA) and 6 with intermediate resistance to vancomycin (VISA). All isolates were screened by PCR for the presence of qacA, B, C, G, H and J. Biocides had MBCs 10-1000-fold lower than the concentration recommended for use by the manufacturer. HA-MRSA isolates developed significantly enhanced tolerance to QACs following repeat exposure to subinhibitory concentrations. Ten HA-MRSA and four VISA isolates carried qacA. Two HA-MRSA isolates, one MSSA isolate and one VISA isolate carried qacC. One VISA isolate carried qacA and qacC. The CA-MRSA isolates did not carry qac genes. qacG, H and J were not detected in any HA-MRSA. Isolates with qac genes had significantly (P < 0.0001) higher MBCs for biocides containing QACs and chlorhexidine gluconate. These biocides induced expression of qac genes when assayed with a luciferase reporter. Biocides commonly used in the hospital environment should be effective against clinical isolates of S. aureus if used at concentrations recommended by the manufacturer. However, isolates have the potential to develop increased tolerance to these agents and the expression of Qac efflux pumps results in isolates with a selective advantage when challenged with biocides containing QACs and chlorhexidine gluconate.


Airway management of lingual tonsillar hypertrophy: A narrative review.
Lingual tonsillar hypertrophy is rarely identified on routine airway assessment but may cause difficulties in airway management. We conducted a narrative review of case reports of lingual tonsillar hypertrophy to examine associated patient factors, success rates of airway management techniques and complications. We searched the literature for anaesthetic management of cases with lingual tonsillar hypertrophy. We found 89 patients in various case reports, from which we derived 92 cases to analyse. 64% of cases were assessed as having a normal airway. Difficult and impossible face mask ventilation occurred in 29.6% and 1.4% of cases, respectively. Difficult intubation and failed intubation occurred in 89.1% and 21.7% of cases, respectively. Multiple attempts (up to six) at intubation were performed, with no successful intubation after the third attempt with direct laryngoscopy. Some 16.5% of patients were woken up and 4.3% required emergency front of neck access. Complications included oesophageal intubation (10.9%), bleeding (9.8%) and severe hypoxia (3.2%). Our findings show that severe cases of lingual hypertrophy may cause an unanticipated difficult airway and serious complications, including hypoxic brain damage and death. A robust airway strategy is required which includes limiting the number of attempts at laryngoscopy, and early priming and performance of emergency front of neck access if required. In patients with known severe lingual tonsillar hypertrophy, awake intubation should be considered.


Does primate motion perception depend on the magnocellular pathway?
This study examined the importance of the primate magnocellular retinocortical pathway in the perception of moving stimuli. A portion of the magnocellular pathway was permanently and selectively interrupted by ibotenic acid injections in the LGN of macaque monkeys. We then tested contrast sensitivity for detecting moving stimuli, as well as two indices of motion perception, contrast sensitivity for opposite direction discrimination and speed difference thresholds, in the affected portion of the visual field. Magnocellular lesions greatly reduced detection contrast sensitivity at high temporal and low spatial frequencies and had a similar effect on contrast sensitivity for opposite direction discrimination under these same stimulus conditions. Consequently, opposite direction discriminations could be made at contrast threshold, suggesting that magnocellular lesions reduced the visibility of stimuli used to test direction perception, but did not act directly on direction perception. Magnocellular lesions also elevated speed difference thresholds under some stimulus conditions. However, this deficit was reduced or eliminated by raising the contrast of the test stimulus. Together, these findings suggest that magnocellular lesions reduce the visibility of stimuli used to test motion perception but that they do not appear to alter motion perception otherwise.


Effect of laminated hydroxyapatite/gelatin nanocomposite scaffold structure on osteogenesis using unrestricted somatic stem cells in rat.
Bone matrix consists of two major phases at the nanoscale: organic and hydroxyapatite. Nanotechnology as a diverse and interdisciplinary area of research has the capacity to revolutionise many areas of applications such as bone tissue engineering. Nanohydroxyapatite/gelatin composite has higher osteoblast attachment and proliferation than micro-sized ones, and shorter culturing period and lower cell seeding density compared to pure gelatin. A nanostructured scaffold was fabricated by three methods for bone repair using nanohydroxyapatite and gelatin as the main components. Its biocompatibility, alizarin red test on the 14th and 21st days, gene expression on the 21st day in in vitro using and histomorphometry after 4 and 8 weeks post-implantation in the rat were investigated. Cultured unrestricted somatic stem cells used for in vitro study showed an excellent level of cell attachment to the scaffold. Cells induced more osteoblast differentiation on the scaffold than in 2D cell culture. Osteoblast differentiation and bone regeneration results of in vitro and in vivo investigation on scaffold were extremely significant, better than control and treatment groups. These effects could be attributed to the shape and size of nanoHA particles and good architecture of the scaffold. The results confirm the feasibility of bone regeneration using synthesised scaffold as a temporary bone substitute.


[Method of calculating self-shielding of the human critical organs in the anthropomorphic phantom].
Proposed is a procedure of calculating the self-screening function of a point on the human body model. The procedure has been developed for the anthropomorphic phantom approved in Russian standard GOST 25645.203-83. The procedure allows extrapolation of the self-shielding calculations to a different body model and a partially protected human body (intra- and extravehicular spacesuit, etc). Results can be useful in precise determination of radiation risks with consideration of the organs self-shielding factor.


Proteomic-based approach for biomarkers discovery in early detection of invasive urothelial carcinoma.
Identification of reliable non-invasive markers for the detection of invasive phenotype of urothelial carcinoma is needed. This study characterizes and compares protein expression profiles of adjacent non-neoplastic urothelium and invasive urothelial carcinoma to identify biomarkers for early detection of de novo bladder cancer. Differences in protein expression between adjacent non-neoplastic and high-grade, stage T4, grade 3 invasive urothelial carcinoma tissues were investigated using 2-DE, MALDI-TOF-MS, and data processing. Ingenuity Pathway Analysis (IPA) was applied to examine the biological mechanisms represented by the altered proteins. The 2-DE of the adjacent non-neoplastic urothelium and invasive urothelial carcinoma showed reproducibly similar proteomic mapping for each group distinguishing adjacent non-neoplastic urothelium from invasive urothelial carcinoma. Twenty-one proteins were altered in expression and one of these proteins, Choroideremia-like protein (CHML) was significantly overexpressed (p<0.005) and therefore was analyzed further using IHC and Western blot. Urothelial carcinoma presented an elevated expression of CHML but not adjacent non-neoplastic or normal bladder tissues. IPA revealed the involvement of CHML in cell morphology, cellular assembly, and organization. Further investigation is warranted to elucidate the biological significance of CHML and to validate its role as a biomarker for early detection of invasive urothelial carcinoma de novo.


The Prevalence of Japanese Outpatients with Hypertension Who Meet the Definition of Treatment Resistant Hypertension and Are Eligible for Enrolment in Clinical Trials of Endovascular Ultrasound Renal Denervation.
Objective A clinical trial (REQUIRE) was started to investigate the use of an ultrasound renal denervation system in the treatment of resistant hypertension (RHT). We analyzed the prevalence of patients who were eligible for inclusion in this cross-sectional study at the time of screening. Methods Nine-hundred ninety-nine consecutive hypertension (HT) patients who were treated in our hospital as outpatients were classified into the following categories: patients treated with at least 3 types of antihypertensive drugs including diuretic agents who were eligible for enrolment in SYMPLICITY HTN-Japan (SH-J) with an office systolic blood pressure (SBP) of ≥160 mmHg, who were ≤80 years of age, and an estimated glomerular filtration rate (eGFR) of ≥45 mL/min/1.73 m2 (RHT-S); and patients who were treated similar medications and who were eligible for enrolment in REQUIRE, with an SBP of ≥150 mmHg, ≤75 years of age, and an eGFR of ≥40 mL/min/1.73 m2 (RHT-R). We investigated the proportion of patients in each category. We also investigated HT patients (1,423 cases) who were enrolled in the Chikushi Anti-Hypertension Trial (CHAT), a research network that includes general practitioners. Results Eleven patients (1.1%) with RHT-S and 18 patients (1.8%) with RHT-R were identified. After the exclusion of patients with secondary HT and a diastolic blood pressure (DBP) of <90 mmHg (applied in REQUIRE), 5 patients (0.5%) with RHT-S and 4 patients (0.4%) with RHT-R remained. In the analysis of the CHAT study, only 2 (0.1%) patients with RHT-R remained. Conclusion The number of eligible patients in the REQUIRE trial was decreased, largely due to the strict age restriction and the new DBP limitation. The prevalence of eligible patients in REQUIRE was estimated to be approximately 0.5 to 0.8 times that in SH-J. Since patient enrollment will be difficult, drastic measures may be required to recruit eligible patients.


Repeated evolution of limblessness and digging heads in worm lizards revealed by DNA from old bones.
The evolutionary relationships of the burrowing amphisbaenians ('worm lizards') have long been controversial for several reasons: the rarity of museum specimens available for study, highly derived morphological conditions that can confound comparative studies and difficulty in obtaining tissues for molecular phylogenetic studies because of their secretive habits in the wild. We present a phylogenetic analysis of two nuclear genes obtained from both fresh tissues and museum specimens of worm lizards. We achieved sufficient taxonomic sampling for analysis by extracting DNA from museum specimens using a modified forensics protocol. Results show the limbless Rhineuridae to be the most basal lineage, whereas the limbed Bipedidae occupy a more derived position as the sister-taxon to a Trogonophidae-Amphisbaenidae clade. This pattern of relationships indicates widespread morphological convergence within the group, including three independent incidences of limb loss. Convergence in skull shape and scalation is also prevalent. Mosaic evolution in the skull versus postcranial skeleton parallels that seen in snake evolution.


Wind-associated detours promote seasonal migratory connectivity in a flapping flying long-distance avian migrant.
It is essential to gain knowledge about the causes and extent of migratory connectivity between stationary periods of migrants to further the understanding of processes affecting populations, and to allow efficient implementation of conservation efforts throughout the annual cycle. Avian migrants likely use optimal routes with respect to mode of locomotion, orientation and migration strategy, influenced by external factors such as wind and topography. In self-powered flapping flying birds, any increases in fuel loads are associated with added flight costs. Energy-minimizing migrants are therefore predicted to trade-off extended detours against reduced travel across ecological barriers with no or limited foraging opportunities. Here, we quantify the extent of detours taken by different populations of European nightjars Caprimulgus europaeus, to test our predictions that they used routes beneficial according to energetic principles and evaluate the effect of route shape on seasonal migratory connectivity. We combined data on birds tracked from breeding sites along a longitudinal gradient from England to Sweden. We analysed the migratory connectivity between breeding and main non-breeding sites, and en route stopover sites just south of the Sahara desert. We quantified each track's route extension relative to the direct route between breeding and wintering sites, respectively, and contrasted it to the potential detour derived from the barrier reduction along the track while accounting for potential wind effects. Nightjars extended their tracks from the direct route between breeding and main non-breeding sites as they crossed the Mediterranean Sea-Sahara desert, the major ecological barrier in the Palaearctic-African migration system. These clockwise detours were small for birds from eastern sites but increased from east to west breeding longitude. Routes of the tracked birds were associated with partial reduction in the barrier crossing resulting in a trade-off between route extension and barrier reduction, as expected in an energy-minimizing migrant. This study demonstrates how the costs of barrier crossings in prevailing winds can disrupt migratory routes towards slightly different goals, and thereby promote migratory connectivity. This is an important link between individual migration strategies in association with an ecological barrier, and both spatially and demographic population patterns.


Diffusion behavior of gap junction hemichannels in living cells.
Due to its non-invasive character, fluorescence correlation spectroscopy (FCS) is particularly suited for the investigation of diffusion behavior of proteins in living cells. In this study we have investigated the diffusion properties of CFP-labeled gap junction hemichannels in the plasma membrane of living HeLa cells. Gap junction hemichannels or connexons are the precursors for the cell-cell- or gap junction channels that form large plaques at the contact areas between two adjacent cells. It has been proposed that new channels are recruited into a gap junction structure from a pool of hemichannels that can freely diffuse over the entire plasma membrane. The statistical approach shows that the geometry of the membrane within the focus is the most important property for the form of the autocorrelation curve and in turn for the determination of the diffusion coefficient. On the other hand binding-unbinding events which lead to anomalous diffusion have only a minor effect to the position and shape of the correlation curve compared to the geometry of the membrane.


Arthrography of the knee. II. Isolated and combined lesions.
The findings in 1 349 abnormal cases out of 2 836 arhtrographies of the knee have been evaluated statistically. Isolated and combined lesions of menisci, medial collateral and cruciate ligaments have been compared. Type and degree of the individual lesion change with increased complexity of combined injuries. A sequential grading for some lesions has been established. A dominant direction of rupture was found differing in themedial and lateral meniscus. Vertical ruptures anf ruptures of the posterior part of the medial meniscus were positively correlated to more complex injuries.


To Mimic Mechanical Properties of the Skin by Inducing Oriented Nanofiber Microstructures in Bottlebrush Cellulose-graft-diblock Copolymer Elastomers.
Skin is a vital biological defense system that protects the body from physical harm with its unique mechanical properties attributed to the hierarchical organization of the protein scaffold. Developing a synthetic skinlike material has aroused great interest; however, replication of the skin's mechanical response, including anisotropic softness and strain-stiffening, is difficult to achieve. Here, to mimic the mechanical behaviors of skin, a reprocessable bottlebrush copolymer elastomer was designed with renewable and rigid cellulose as backbones; meanwhile, poly(n-butyl acrylate)-b-poly(methyl methacrylate) (PBA-b-PMMA) diblocks were designed as the grafted side chains. The so-made elastomers were subjected to a step-cyclic tensile deformation, by which the internal structures became oriented nanofibers and endowed stress-strain behaviors pretty much similar to those of the real skin. Overall, our research work currently undertaken would be of great importance in the development of a series of biomimetic skinlike polymer materials.


MRGPRX2 is essential for sinomenine hydrochloride induced anaphylactoid reactions.
Mast cells are unique immunocytes that function as sentinel cells in host defense reactions such as immediate hypersensitivity responses and anaphylactic responses. The mast cell specific receptor MRGPRX2 (Mas-related G protein-coupled receptor X2) triggers mast cell degranulation-a key process in anaphylactic reactions. We sought to better understand anaphylactic reaction induced by sinomenine hydrochloride (SH). MRGPRX2-related pseudo-allergic reactions induced by SH were investigated using the hindpaw swelling and extravasation assay in vivo and mast cell degranulation assays in vitro. MrgprB2 knockout mice exhibit a reduced SH-induced inflammation effect. Furthermore, MRGPRX2 (the orthologous gene of MrgprB2) related human mast cells are activated by SH in a dose-dependent manner; however, MRGPRX2 knockdown mast cells showed reduced degranulation. The results showed a kind of mechanism that SH-induced anaphylactoid reactions were mediated by MRGPRX2 via activating PLC molecular signaling pathways to provoke mast cells Ca2+ mobilization and degranulation.


Generation from tumor-bearing mice of lymphocytes with in vivo therapeutic efficacy.
Systemic transfer of sensitized lymphocytes can effectively mediate the regression of established tumors. However, virtually all prior experimental applications of this approach have utilized lymphocytes from animals that have been immunized to reject tumor challenge. A similar source of cells is not available in the human. With the use of a weakly immunogenic murine tumor, MCA 105, we demonstrate here that following in vitro sensitization (IVS) with viable tumor cells and interleukin 2, the nontherapeutic lymphoid cells from mice bearing a progressively growing tumor acquired antitumor reactivity capable of mediating the regression of established pulmonary metastases. Although the IVS system induced nonspecific lymphokine-activated killer-like cytotoxic activity from lymphoid cells of normal as well as tumor-bearing mice, therapeutically active cells could only be generated from cultures initiated with lymphoid cells from tumor-bearing animals, indicating that the IVS was a secondary in vitro immune response. Without other treatment, the IVS cells could mediate antitumor effects. However, low doses of exogenous interleukin 2 administration could enhance their therapeutic efficacy. By in vivo T cell subset depletion with monoclonal antibodies, the primary effector cells were identified as belonging to cytotoxic/suppressor T cell lineage expressing the Lyt-2 phenotype. In addition, these therapeutic effector cells could be further expanded in numbers in vitro with continuous stimulation by tumor cells in the presence of interleukin 2. Compared to the number of cells initiating the culture, as many as 126 times the number of cells were obtained after 9 days of IVS followed by in vitro expansion for an additional 5 days. Studies on the kinetics of the occurrence of the pre-effector lymphocytes during tumor growth revealed that they were readily obtained from draining lymph nodes of mice with a broad range of tumor burdens as well as durations of tumor growth. The ability to generate and expand, in vitro, therapeutically active lymphocytes from tumor-bearing hosts has important implications for cellular therapy of human cancers.


Specific binding of modified ZD6474 (Vandetanib) monomer and its dimer with VEGF receptor-2.
Tumor angiogenesis is an important component of cancer biology driven in part by the hypothesis that tumor vessel growth is a necessary requirement for tumor growth. Angiogenesis does not only depend on endothelial cell invasion and proliferation, it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF). Vandetanib, also known as ZD6474, is an orally bioavailable small molecule tyrosine kinase inhibitor of multiple growth factors that is an antagonist of the vascular endothelial growth factor receptor-2 (VEGFR-2). ZD6474 was purchased and modified to add a fluorescent dye (6-FAM). A linker was used to couple the ZD6474 monomer to create dimers, and similarly linked to FAM fluorescent dye. The two compounds were compared using human endothelial cell (HUVECs) and the cancer cell lines U-87-MG and MDA-MB-231. We compared cellular uptake and binding specificity, as well as effects on cellular viability and angiogenesis in a series of in vitro and in vivo studies. ZD6474 dimer demonstrated improved uptake in HUVECs and other VEGFR expressing cells over ZD6474 monomer in vitro. Therapeutic effects were mixed, with in vitro studies showing the dimer having the same or weaker effects compared to the monomer, while an in vivo study using pseudotumors (matrigel plug assay) seemed to indicate stronger effects could be obtained from the dimer. Finally, in biodistribution study in a xenograft tumor model, the dimer accumulated 20× greater concentration in the tumor than in the liver, spleen, or kidneys, and also 20× greater than the accumulation of the monomer or the dye alone, all at 24 h following compound injection. This study provides a rationale for the evaluation of dimeric ZD6474 as a potent imaging agent of angiogenic activity in vivo.


Sub-regional anterior cingulate cortex functional connectivity revealed default network subsystem dysfunction in patients with major depressive disorder.
Major depressive disorder (MDD) is a prevalent mental disorder characterized by impairments in affect, behaviour and cognition. Previous studies have indicated that the anterior cingulate cortex (ACC) may play an essential role in the pathophysiology of depression. In this study, we systematically identified changes in functional connectivity (FC) for ACC subdivisions that manifest in MDD and further investigated the relationship between these changes and the clinical symptoms of depression. Sub-regional ACC FC was estimated in 41 first-episode medication-naïve MDD patients compared to 43 healthy controls. The relationships between depressive symptom severity and aberrant FC of ACC subdivisions were investigated. In addition, we conducted a meta-analysis to generate the distributions of MDD-related abnormal regions from previously reported results and compared them to FC deficits revealed in this study. In MDD patients, the subgenual and perigenual ACC demonstrated decreased FC with the posterior regions of the default network (DN), including the posterior inferior parietal lobule and posterior cingulate cortex. FC of these regions was negatively associated with the Automatic Thoughts Questionnaire scores and largely overlapped with previously reported abnormal regions. In addition, reduced FC between the caudal ACC and precuneus was negatively correlated with the Hamilton Anxiety Scale scores. We also found increased FC between the rostral ACC and dorsal medial prefrontal cortex. Our findings confirmed that functional interaction changes in different ACC sub-regions are specific and associated with distinct symptoms of depression. Our findings provide new insights into the role of ACC sub-regions and DN in the pathophysiology of MDD.


Twelve Tips for using Learning Curves in Health Professions Education Research.
Learning curves can be used to design, implement, and evaluate educational interventions. Attention to key aspects of the method can improve the fidelity of this representation of learning as well as its suitability for education and research purposes. This paper addresses when to use a learning curve, which graphical properties to consider, how to use learning curves quantitatively, and how to use observed thresholds to communicate meaning. We also address the associated ethics and policy considerations. We conclude with a best practices checklist for both educators and researchers seeking to use learning curves in their work.


Feasibility of whole RNA sequencing from single-cell mRNA amplification.
Single-cell sampling with RNA-seq analysis plays an important role in reference laboratory; cytogenomic diagnosis for specimens on glass-slides or rare cells in circulating blood for tumor and genetic diseases; measurement of sensitivity and specificity in tumor-tissue genomic analysis with mixed-cells; mechanism analysis of differentiation and proliferation of cancer stem cell for academic purpose. Our single- cell RNA-seq technique shows that fragments were 250-450 bp after fragmentation, amplification, and adapter addition. There were 11.6 million reads mapped in raw sequencing reads (19.6 million). The numbers of mapped genes, mapped transcripts, and mapped exons were 31,332, 41,210, and 85,786, respectively. All QC results demonstrated that RNA-seq techniques could be used for single-cell genomic performance. Analysis of the mapped genes showed that the number of genes mapped by RNA-seq (6767 genes) was much higher than that of differential display (288 libraries) among similar specimens which we have developed and published. The single-cell RNA-seq can detect gene splicing using different subtype TGF-beta analysis. The results from using Q-rtPCR tests demonstrated that sensitivity is 76% and specificity is 55% from single-cell RNA-seq technique with some gene expression missing (2/8 genes). However, it will be feasible to use RNA-seq techniques to contribute to genomic medicine at single-cell level.


Effect-Based Identification of Hazardous Antibiotic Transformation Products after Water Chlorination.
Antibiotic transformation products (TPs) generated during water treatment can be considered as an environmental concern, since they can retain part of the bioactivity of the parent compound. Effect-directed analysis (EDA) was applied for the identification of bioactive intermediates of azithromycin (AZI) and ciprofloxacin (CFC) after water chlorination. Fractionation of samples allowed the identification of bioactive intermediates by measuring the antibiotic activity and acute toxicity, combined with an automated suspect screening approach for chemical analysis. While the removal of AZI was in line with the decrease of bioactivity in chlorinated samples, an increase of bioactivity after complete removal of CFC was observed (at >0.5 mgCl2/L). Principal component analysis (PCA) revealed that some of the CFC intermediates could contribute to the overall toxicity of the chlorinated samples. Fractionation of bioactive samples identified that the chlorinated TP296 (generated from the destruction of the CFC piperazine ring) maintained 41%, 44%, and 30% of the antibiotic activity of the parent compound in chlorinated samples at 2.0, 3.0, and 4.0 mgCl2/L, respectively. These results indicate the spectrum of antibacterial activity can be altered by controlling the chemical substituents and configuration of the CFC structure with chlorine. On the other hand, the potential presence of volatile DBPs and fractionation losses do not allow for tentative confirmation of the main intermediates contributing to the acute toxic effects measured in chlorinated samples. Our results encourage further development of new and advanced methodologies to study the bioactivity of isolated unknown TPs to understand their hazardous effects in treated effluents.


Impact of input data (in)accuracy on overestimation of visible area in digital viewshed models.
Viewshed analysis is a GIS tool in standard use for more than two decades to perform numerous scientific and practical tasks. The reliability of the resulting viewshed model depends on the computational algorithm and the quality of the input digital surface model (DSM). Although many studies have dealt with improving viewshed algorithms, only a few studies have focused on the effect of the spatial accuracy of input data. Here, we compare simple binary viewshed models based on DSMs having varying levels of detail with viewshed models created using LiDAR DSM. The compared DSMs were calculated as the sums of digital terrain models (DTMs) and layers of forests and buildings with expertly assigned heights. Both elevation data and the visibility obstacle layers were prepared using digital vector maps differing in scale (1:5,000, 1:25,000, and 1:500,000) as well as using a combination of a LiDAR DTM with objects vectorized on an orthophotomap. All analyses were performed for 104 sample locations of 5 km2, covering areas from lowlands to mountains and including farmlands as well as afforested landscapes. We worked with two observer point heights, the first (1.8 m) simulating observation by a person standing on the ground and the second (80 m) as observation from high structures such as wind turbines, and with five estimates of forest heights (15, 20, 25, 30, and 35 m). At all height estimations, all of the vector-based DSMs used resulted in overestimations of visible areas considerably greater than those from the LiDAR DSM. In comparison to the effect from input data scale, the effect from object height estimation was shown to be secondary.


Cardiovascular implications of abdominal compartment syndrome.
Cardiovascular dysfunction and failure are commonly encountered in the patient with intra-abdominal hypertension or abdominal compartment syndrome. Accurate assessment and optimization of preload, contractility, and afterload, in conjunction with appropriate goal-directed resuscitation and abdominal decompression when indicated, are essential to restoring end-organ perfusion and maximizing patient survival. The validity of traditional hemodynamic resuscitation endpoints, such as pulmonary artery occlusion pressure and central venous pressure, must be reconsidered in the patient with intra-abdominal hypertension as these pressure-based estimates of intravascular volume have significant limitations in patients with elevated intra-abdominal pressure. If such limitations are not recognized, misinterpretation of the patient's cardiac status is likely, resulting in inappropriate and potentially detrimental therapy. Appropriate fluid administration is mandatory as under-resuscitation leads to organ failure and over-resuscitation the development of secondary abdominal compartment syndrome, both of which are associated with increased morbidity and mortality. Volumetric monitoring techniques have been proven to be superior to traditional intra-cardiac filling pressures in directing the appropriate resuscitation of this patient population. Calculation of the "abdominal perfusion pressure", defined as mean arterial pressure minus intra-abdominal pressure, has been shown to be a beneficial resuscitation endpoint as it assesses not only the severity of the patient's intra-abdominal hypertension, but also the adequacy of abdominal blood flow. Application of a goal-directed resuscitation strategy, including abdominal decompression when indicated, improves cardiac function, reverses end-organ failure, and minimizes intra-abdominal hypertension-related patient morbidity and mortality.


Bone marrow norepinephrine mediates development of functionally different macrophages after thermal injury and sepsis.
We sought to determine the influence of thermal (burn) injury with sepsis and norepinephrine on the clonogenic potential and functional cytokine response to lipopolysaccharide (LPS) stimulation in nonmyeloid committed (CD117) and myeloid committed (ER-MP12) bone marrow progenitor cells. We have previously demonstrated that norepinephrine stimulated myelopoiesis after burn injury and sepsis, but the site of this stimulation in monocyte development is unknown. In the present study the influence of norepinephrine on the developmental hierarchy of bone marrow cells after thermal injury and sepsis was determined by assessing the clonogenic potential and LPS-stimulated cytokine responses of mature macrophages derived from CD117 and ER-MP12 bone marrow progenitor cells. Tissue and bone marrow norepinephrine content was ablated by chemical sympathectomy with 6-hydroxydopamine treatment. CD117 and ER-MP12 bone marrow cells were isolated using antibody-linked magnetic microbeads. Clonogenic potential in response to colony-stimulating factors was determined. Both progenitor cell types were differentiated to mature macrophages in vitro and tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 cytokine responses to LPS provocation were determined. The macrophage- and granulocyte-macrophage colony-stimulating factor responsive clonogenic potential was increased with burn sepsis, suggesting an expansion of both progenitor populations. Such increases were greatly reduced with prior depletion of norepinephrine. TNF-alpha and IL-6 cytokine responses to LPS were markedly influenced by the specific progenitor cells involved as well as the injury conditions and the status of norepinephrine prior to injury. In burn sepsis the depletion of norepinephrine resulted in a dramatic decrease in both IL-6 and TNF-alpha production by both progenitor-derived macrophages. Depletion of norepinephrine attenuated burn and burn sepsis-induced bone marrow progenitor clonal growth in response to macrophage- and granulocyte-macrophage colony-stimulating factor. Functional phenotypes of bone marrow progenitor-derived macrophages are greatly influenced by norepinephrine and the milieu created by thermal injury and sepsis.


High-Performance Acellular Tissue Scaffold Combined with Hydrogel Polymers for Regenerative Medicine.
Decellularization of tissues provides extracellular matrix (ECM) scaffolds for regeneration therapy and an experimental model to understand ECM and cellular interactions. However, decellularization often causes microstructure disintegration and reduction of physical strength, which greatly limits the use of this technique in soft organs or in applications that require maintenance of physical strength. Here, we present a new tissue decellularization procedure, namely CASPER (Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine), which includes infusion and hydrogel polymerization steps prior to robust chemical decellularization treatments. Polymerized hydrogels serve to prevent excessive damage to the ECM while maintaining the sophisticated structures and biological activities of ECM components in various organs, including soft tissues such as brains and embryos. CASPERized tissues were successfully recellularized to stimulate a tissue-regeneration-like process after implantation without signs of pathological inflammation or fibrosis in vivo, suggesting that CASPERized tissues can be used for monitoring cell-ECM interactions and for surrogate organ transplantation.


Production of reactive oxygen species and 8-hydroxy-2'deoxyguanosine in KB cells co-exposed to benzo[a]pyrene and UV-A radiation.
Previous studies have shown that ultraviolet (UV) A light and the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) can synergistically enhance the formation of 8-hydroxy-2'deoxyguanosine (8-OHdG) in living cells. It has been postulated that the underlying mechanism is production of reactive oxygen species (ROS) via photosensitization, but direct evidence supporting this hypothesis has been lacking. This study examined intracellular ROS production in living cells co-exposed to UV-A and BaP as well as the relationship between intracellular production of ROS and formation of 8-OHdG. KB cells were exposed to BaP for 24 h, followed by exposure to UV-A (365 nm) or UV-B (312 nm). The levels of intracellular ROS were directly measured by use of the fluorescent probe dihydrorhodamine 123 (DHR-123) in flow cytometry. Levels of 8-OHdG were measured by high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD). The results demonstrated that UV-B itself induced a much greater level of intracellular ROS than did UV-A alone under the same dose of energy (0.10 mW/cm(2), 20 min). The presence of BaP (13.3 microM) substantially increased ROS production in UV-A-treated cells (2.9-fold), but only slightly enhanced ROS production in UV-B-treated cells (1.3-fold). These results show that BaP acts mainly as a photosensitizer of UV-A, but not UV-B. Furthermore, greater intracellular ROS production was proportional to both BaP concentration and UV-A dosage. There was a linear relationship between ROS production and 8-OHdG formation in cells co-exposed to BaP and UV-A. Results of this study suggest that UV-A and BaP act synergistically to enhance ROS production and formation of 8-OHdG, resulting in increased DNA damage.


Microsatellite loci for the small hive beetle, Aethina tumida, a nest parasite of honey bees.
Aethina tumida, a beetle parasite of honey bee colonies, has recently and dramatically expanded its range and now parasitizes honey bees on three continents. Polymorphic microsatellite loci for this beetle species will help map this continuing range expansion, and will also prove useful for exploring the mating system and local gene flow patterns for this important parasite. We describe 15 loci that are polymorphic in both the native and introduced ranges of this species, showing from two to 22 alleles.


Corneal epithelial wound healing.
One of the important functions of the cornea is to maintain normal vision by refracting light onto the lens and retina. This property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Epithelial renewal is essential because it enables this tissue to act as a barrier that protects the corneal interior from becoming infected by noxious environmental agents. The renewal process also maintains the smooth optical surface of the cornea. This rate of renewal is closely maintained by an integrated balance between the processes of corneal epithelial proliferation, differentiation, and cell death. Attempts to understand this complex cascade make it evident that the appropriate integration and coordination of corneal epithelial renewal depends on the actions of a myriad of cytokines. We have attempted in this review to collate the receptor and cell signaling events and cytokine studies that are responsible for mediating corneal wound healing.


The nature of antigen in the eye has a profound effect on the cytokine milieu and resultant immune response.
The eye is endowed with a number of mechanisms that protect it from immune-mediated injury. One such mechanism, termed anterior chamber-associated immune deviation (ACAID), evokes the antigen-specific, systemic down-regulation of Th1 responses to antigen inoculated into the anterior chamber of the eye. ACAID has been correlated with the selective production of IL-10 by the antigen-presenting cells (APC) and the development of a cross-regulatory Th2-like response. A small subset of antigens do not induce ACAID, but instead provoke IL-12 and normal Th1 immunity. Remarkably, all soluble antigens tested are capable of inducing ACAID; only cell-associated antigens do not induce ACAID. We hypothesized that the nature of antigen plays a decisive role in the resultant immune response. This hypothesis was tested with two well-characterized antigens, ovalbumin (OVA) and SV40 large T antigen (SV40 Lg T Ag). The soluble forms of OVA and SV40 Lg T Ag induced ACAID in both in vivo and in vitro models of the eye. In contrast, the particulate forms of these antigens, i.e. OVA passively absorbed onto inert latex beads (OVA-latex) and SV40 Lg T Ag expressed in two different cell lines, 99E1 and SV-T2, did not induce ACAID in either in vivo or in vitro models of the eye. In addition, the cytokine profiles of ocular APC pulsed with OVA or OVA-latex showed that soluble OVA induced the production of IL-10, whereas OVA-latex induced the production of IL-12. These data suggest that the nature of the antigen in the eye, whether soluble or particulate, is a crucial determinant in the resultant immune response. Moreover, they suggest a mechanism in which soluble antigens preferentially induce the release of ACAID-inducing IL-10 whereas particulate antigens preferentially induce the release of Th1-inducing IL-12 by responding APC.


Solid-Phase Assembly of Multienzyme Systems into Artificial Cellulosomes.
We herein describe a bioinspired solid-phase assembly of a multienzyme system scaffolded on an artificial cellulosome. An alcohol dehydrogenase and an ω-transaminase were fused to cohesin and dockerin domains to drive their sequential and ordered coimmobilization on agarose porous microbeads. The resulting immobilized scaffolded enzymatic cellulosome was characterized through quartz crystal microbalance with dissipation and confocal laser scanning microscopy to demonstrate that both enzymes interact with each other and physically colocalize within the microbeads. Finally, the assembled multifunctional heterogeneous biocatalyst was tested for the one-pot conversion of alcohols into amines. By using the physically colocalized enzymatic system confined into porous microbeads, the yield of the corresponding amine was 1.3 and 10 times higher than the spatially segregated immobilized system and the free enzymes, respectively. This work establishes the basis of a new concept to organize multienzyme systems at the nanoscale within solid and porous immobilization carriers.


Apoptosis in primary lymphoid organs with aging.
Age-associated changes in the immune system are responsible for an increased likelihood of infection, autoimmune diseases, and cancer in the elderly. Immunosenescence is characterized by reduced levels of the peripheral naive T cell pool derived from thymus and the loss of immature B lineage cells in the bone marrow. Primary lymphoid organs, i.e., bone marrow and thymus, exhibit a loss of cellularity with age, which is especially dramatic in the thymus. A summary of major changes associated with aging in primary lymphoid organs is described in this article. The participation of apoptosis in cell loss in the immune system, a change associated with age, as well as a description of molecular machinery involved, is presented. Finally, the involvement of different hormonal and non-hormonal agents in counteracting apoptosis in thymus and bone marrow during aging is explained. Here, we underlie the important role of glucocorticoids as immunodepressors and melatonin as an immunostimulatory agent.


Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system.
We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.


Real-time signal processing for fetal heart rate monitoring.
An algorithm based on digital filtering, adaptive thresholding, statistical properties in the time domain, and differencing of local maxima and minima has been developed for the simultaneous measurement of the fetal and maternal heart rates from the maternal abdominal electrocardiogram during pregnancy and labor for ambulatory monitoring. A microcontroller-based system has been used to implement the algorithm in real-time. A Doppler ultrasound fetal monitor was used for statistical comparison on five volunteers with low risk pregnancies, between 35 and 40 weeks of gestation. Results showed an average percent root mean square difference of 5.32% and linear correlation coefficient from 0.84 to 0.93. The fetal heart rate curves remained inside a +/- 5-beats-per-minute limit relative to the reference ultrasound method for 84.1% of the time.


Prostate cancer genetic risk and associated aggressive disease in men of African ancestry.
African ancestry is a significant risk factor for prostate cancer and advanced disease. Yet, genetic studies have largely been conducted outside the context of Sub-Saharan Africa, identifying 278 common risk variants contributing to a multiethnic polygenic risk score, with rare variants focused on a panel of roughly 20 pathogenic genes. Based on this knowledge, we are unable to determine polygenic risk or differentiate prostate cancer status interrogating whole genome data for 113 Black South African men. To further assess for potentially functional common and rare variant associations, here we interrogate 247,780 exomic variants for 798 Black South African men using a case versus control or aggressive versus non-aggressive study design. Notable genes of interest include HCP5, RFX6 and H3C1 for risk, and MKI67 and KLF5 for aggressive disease. Our study highlights the need for further inclusion across the African diaspora to establish African-relevant risk models aimed at reducing prostate cancer health disparities.


Psychometrics, Reliability, and Validity of a Wraparound Team Observation Measure.
Wraparound is a widely-implemented team-based care coordination process for youth with serious emotional and behavioral needs. Wraparound has a positive evidence base; however, research has shown inconsistency in the quality of its implementation that can reduce its effectiveness. The current paper presents results of three studies used to examine psychometrics, reliability, and validity of a measure of wraparound fidelity as assessed during team meetings called the Team Observation Measure (TOM). Analysis of TOM results from 1,078 team observations across 59 sites found good overall internal consistency (alpha = .80), but constrained variability, with the average team rated as having 78% of indicators of model adherent wraparound present, 11% absent, and 11% not applicable. A study of N=23 pairs of raters found a pooled Kappa statistic of .733, indicating substantial inter-rater reliability. Higher agreement was found between external evaluators than for pairs of raters that included an external evaluator and an internal rater (e.g., supervisor or coach). A validity study found no correlation between the TOM and an alternate fidelity instrument, the Wraparound Fidelity Index (WFI), at the team level. However, positive correlations between mean program-level TOM and WFI scores provide support for TOM validity as a summative assessment of site- or program-level fidelity. Implications for TOM users, measure refinement, and future research are discussed.


Surgical approaches to total hip arthroplasty.
Surgical exposure of the hip for trauma, infection, or reconstruction can be adequately accomplished through a variety of surgical approaches. This article describes in detail five classic approaches to the hip: Smith-Petersen (anterior), Watson-Jones (anterolateral), Hardinge (direct lateral), transtrochanteric, and posterolateral. In addition, recently described mini-incision posterior and anterior approaches are outlined.


[Mothers Talk. Part Unless Told of Voluntary Abortion].
The aim of the study was to investigate the experiences linked to the post-abortion syndrome in mothers who have had a voluntary abortion. A phenomenological qualitative approach to collect the experiences of mothers who had voluntarily interrupted their pregnancy was used. The research technique was the semistructured interviews with women who had contacted different association's help of the Murcia region for support after experiencing symptoms consistent with post-abortion syndrome. The testimonies show feminist or utilitarian arguments to justify the decision to abort, they talk about a system, to some extent, mercantilist that has no real intention of giving real life choices. Experience shows that far from lived as an act of female freedom is experienced traumatically, developed symptoms following the sense of loss. In this context, the resource spiritual becomes the best tool to expiate guilt. The experience of abortion does not improve the lives of women; far from it is a trauma that can be avoided with proper advice to avoid the tragedy of abortion.


[Arterial hypertension and cerebrovascular pathology. Current status of the problem].
In the process of development and progression of arterial hypertension hypertensive cerebral angiopathy is formed - that is a complex of intricate primarily destructive (acute and recurring) and secondary reparative changes and adaptive processes occurring on all structural and functional levels of cerebral vascular system. Arterial hypertension in patients with cerebrovascular diseases should be treated. Because of heterogeneity of etiology and pathogenetic mechanisms of stroke detailed assessment of the state of cerebral vessels, blood flow, circulatory and metabolic cerebral reserve is a prerequisite of effective and safe antihypertensive therapy, prevention of stroke and other complications. This excludes generalized (leveling-off) approach to treatment and prevention of various types and subtypes of stroke. A notion that hypertension is a most important, independent controllable risk factor of cerebrovascular diseases remains definitely valid. However it is far from being a single cause of stroke.


Glucocorticoids differentially increase nerve growth factor and basic fibroblast growth factor expression in the rat brain.
Adrenocorticotropin hormone (ACTH) and adrenal steroids may influence trophic processes operative in neuronal plasticity. Because nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) participate in neuronal trophism, we have investigated whether adrenal steroids induce the expression of these two trophic factors in the rat brain. The systemic administration of dexamethasone (DEX) elicited a rapid (within 3 hr) and sustained accumulation of bFGF and NGF mRNA in the cerebral cortex and hippocampus. Regional studies showed that DEX increases bFGF but not NGF mRNA in the cerebellum, striatum, and hypothalamus. In situ hybridization studies revealed that DEX increases NGF mRNA in superficial layers of the cerebral cortex and in the dentate gyrus of the hippocampus, and bFGF mRNA throughout the brain, suggesting that DEX induces NGF mRNA in neurons and bFGF in glial cells. ACTH administered systemically elicited a temporal and regional induction in NGF and bFGF mRNA similar to that obtained with DEX. Increases in NGF and bFGF mRNAs were also observed after administration of corticosterone and, albeit to a lesser extent, aldosterone, suggesting that the pituitary-adrenocortical axis plays an important role in the regulation of NGF and bFGF expression in the brain. Our data suggest that NGF and bFGF represent a link by which the adrenal cortical system can exert trophic action on the CNS.


Calibration of a comprehensive predictive model for the development of proximal junctional kyphosis and failure in adult spinal deformity patients with consideration of contemporary goals and techniques.
The objective of this study was to calibrate an updated predictive model incorporating novel clinical, radiographic, and prophylactic measures to assess the risk of proximal junctional kyphosis (PJK) and failure (PJF). Operative patients with adult spinal deformity (ASD) and baseline and 2-year postoperative data were included. PJK was defined as ≥ 10° in sagittal Cobb angle between the inferior uppermost instrumented vertebra (UIV) endplate and superior endplate of the UIV + 2 vertebrae. PJF was radiographically defined as a proximal junctional sagittal Cobb angle ≥ 15° with the presence of structural failure and/or mechanical instability, or PJK with reoperation. Backstep conditional binary supervised learning models assessed baseline demographic, clinical, and surgical information to predict the occurrence of PJK and PJF. Internal cross validation of the model was performed via a 70%/30% cohort split. Conditional inference tree analysis determined thresholds at an alpha level of 0.05. Seven hundred seventy-nine patients with ASD (mean 59.87 ± 14.24 years, 78% female, mean BMI 27.78 ± 6.02 kg/m2, mean Charlson Comorbidity Index 1.74 ± 1.71) were included. PJK developed in 50.2% of patients, and 10.5% developed PJF by their last recorded visit. The six most significant demographic, radiographic, surgical, and postoperative predictors of PJK/PJF were baseline age ≥ 74 years, baseline sagittal age-adjusted score (SAAS) T1 pelvic angle modifier > 1, baseline SAAS pelvic tilt modifier > 0, levels fused > 10, nonuse of prophylaxis measures, and 6-week SAAS pelvic incidence minus lumbar lordosis modifier > 1 (all p < 0.015). Overall, the model was deemed significant (p < 0.001), and internally validated receiver operating characteristic analysis returned an area under the curve of 0.923, indicating robust model fit. PJK and PJF remain critical concerns in ASD surgery, and efforts to reduce the occurrence of PJK and PJF have resulted in the development of novel prophylactic techniques and enhanced clinical and radiographic selection criteria. This study demonstrates a validated model incorporating such techniques that may allow for the prediction of clinically significant PJK and PJF, and thus assist in optimizing patient selection, enhancing intraoperative decision making, and reducing postoperative complications in ASD surgery.


Isolation and characterization of Tn5 insertion mutations in the lexA gene of Escherichia coli.
A Mu d(Ap lac)-generated fusion of lacZ to dinD, a gene induced by DNA damage, was used to isolate Tn5 insertion mutations that affect the regulation of the SOS responses. Three mutants were obtained that contained Tn5 insertions genetically linked to the lexA gene and had properties that suggested the mutants were deficient in lexA expression. The lexA protein has been shown to function as the repressor for genes involved in the SOS responses. By Southern blotting experiments, the three Tn5 insertions were physically mapped to distinct locations within the coding region of the lexA gene. The introduction of these mutations in six strains carrying lacZ fusions to different damage-inducible genes resulted in high expression of beta-galactosidase in all but one of the strains. In the dinF fusion strain, lacZ expression was reduced below that seen in a lexA+ background. Physical mapping studies of the dinF locus gave results consistent with the notion that dinF is part of the lexA transcription unit and that a lexA::Tn5 mutation has a polar effect on dinF expression. With certain din-lac fusion strains, a correlation was seen between the amount of beta-galactosidase production and the location of the particular Tn5 insertion within the lexA gene.


[Closed-loop insulin therapy in type 1 diabetes after age 60].
The closed-loop insulin therapy system has become an indispensable tool in the management of type 1 diabetes. This technological feat improves glycemic control while reducing the mental burden on patients. In an exploratory study, we sought to determine whether older patients, who may be less familiar with new technologies, derive the same benefits as others. Is a lack of digital skills an obstacle to improving patients' daily lives?


A lay epidemiological study on coexistent stress in hypertension: Its prevalence, risk factors, and implications in patients' lives.
Hypertension poses a global challenge in terms of morbidity and mortality. Worldwide prevalence of hypertension is over 40%. Management of hypertension targets blood pressure control to prevent disease complications. Though stress and hypertension are closely related, stress management is often overlooked in the treatment of hypertension. (1) To estimate the prevalence of stress in hypertensive patients and (2) to study the associated risk factors of stress and its implications in disease management. It was a hospital-based, cross-sectional study done in Western India for 1 year. Data were collected from 400 hypertensive patients attending the selected health institutions using a pretested questionnaire. Chi-square tests were done using Medcalc 10.4.8.0. The prevalence of stress in hypertensive patients was found to be 84.3%. Only 2.4% of these patients sought help from any health professional for stress. The most common stressors found in the patients were financial dependence on others, living in rented house, having a daughter of marriageable age because of associated dowry, death of a loved one, sleep-related problem, and owing a debt among others. Significant statistical association (P < 0.05) of stress was observed with the type of family and socioeconomic status. A highly significant association (P < 0.001) of stress with religion and residential area (whether urban non-slum, slum, or rural) was observed. Stress in individuals leads to poorer compliance with treatment and blood pressure control. Coexistent stress should be diagnosed and managed in patients of hypertension for proper disease management and control.


Circadian disruption and health: Shift work as a harbinger of the toll taken by electric lighting.
Electric light is one of the signature inventions of human beings. A problem, however, is that electric light can confuse our endogenous circadian rhythmicity. It has now become apparent that circadian biology is fundamental to the functioning and adaptation of almost all life forms. In the modern world, everyone is exposed to electric light during the day and night, and thereby can experience some level of circadian disruption. Perhaps as a canary in the coal mine, study of people whose work hours include nighttime (shift workers) is beginning to yield insights on the adverse health effects of circadian disruption from electric light.


Content validity of the Female Sexual Function Index (FSFI) in pre- and postmenopausal women with hypoactive sexual desire disorder.
The Female Sexual Function Index (FSFI) has consistently been shown to have discriminant validity, test-retest reliability, and internal consistency as a measure of female sexual function. However, the content validity (relevance, clarity, comprehensiveness) of the instrument in women with hypoactive sexual desire disorder (HSDD) must also be established. The aim of this study were to assess the content validity of the FSFI, specifically the FSFI desire domain, in pre- and postmenopausal women with HSDD. Two single-visit content validation studies were conducted in the United States. Eligible premenopausal (both studies) and postmenopausal (second study only) women with HSDD completed the FSFI followed by one-on-one, face-to-face cognitive debriefing interviews including open-ended questions to capture information on their perceptions of the instrument. Information on women's experiences of decreased sexual desire was also captured. The main outcome measures of this study were the women's ratings of the clarity, ease of understanding, comprehensiveness, and relevance of the 19 items of the FSFI. Interviews with 15 premenopausal women (first study), and 30 pre- and 31 postmenopausal women (second study), were analyzed. Across the whole sample, most women (80-100%) found every item of the FSFI clear and easy to understand. The majority (53-70%) felt that the FSFI captured all their feelings about decreased sexual desire and other sexual problems, and most (84-90%) indicated that additional questions were unnecessary. Most women in both studies (93-100%) reported that the two items comprising the FSFI desire domain were clear, easy to understand, and were relevant to them. The majority of women thought that a recall period of ≥7 days is most relevant for recall of their sexual desire. These studies establish the content validity of the FSFI in pre- and postmenopausal women with HSDD, supporting the use of this instrument as a measure of sexual function in women with this condition.


Healthy hire effect, job selection and inhalation exposure among young adults with asthma.
The aim of the present study was to assess whether asthma onset prior to entering the workforce influences whether a person holds a subsequent job with asthma-related inhalation exposures. The data of 19,784 adults from the European Community Respiratory Health Survey were analysed. For each respondent, a current or previously held job was linked to a job exposure matrix assigning high, low or no exposure to dust, gases or fumes. Jobs were also categorised according to the risk of exposures related to occupational asthma. Associations between asthma and subsequent occupational exposures were assessed using logistic regression models, with a random intercept for study centre and fixed adjustment for age, sex, type of study sample and smoking status. Of the respondents, 8% (n = 1,619) reported asthma with onset before completion of full-time education. This population was at decreased risk of having a job with high (odds ratio 0.79; 95% confidence interval 0.68-0.92) or low (0.91; 0.80-1.03) exposure to dust, gases or fumes. The associations were consistent across exposure types (dusts, gases or fumes) and for jobs with a high risk of occupational asthma. Adults with asthma onset prior to entering the workforce may be less likely to hold jobs involving inhalation exposures.


The slippery slope: how small ethical transgressions pave the way for larger future transgressions.
Many recent corporate scandals have been described as resulting from a slippery slope in which a series of small infractions gradually increased over time (e.g., McLean & Elkind, 2003). However, behavioral ethics research has rarely considered how unethical behavior unfolds over time. In this study, we draw on theories of self-regulation to examine whether individuals engage in a slippery slope of increasingly unethical behavior. First, we extend Bandura's (1991, 1999) social-cognitive theory by demonstrating how the mechanism of moral disengagement can reduce ethicality over a series of gradually increasing indiscretions. Second, we draw from recent research connecting regulatory focus theory and behavioral ethics (Gino & Margolis, 2011) to demonstrate that inducing a prevention focus moderates this mediated relationship by reducing one's propensity to slide down the slippery slope. We find support for the developed model across 4 multiround studies.


Nonprogressive and progressive primate immunodeficiency lentivirus infections.
Natural hosts for simian immunodeficiency virus (SIV) can be, and are often naturally, infected with species-specific SIVs, but do not develop acquired immunodeficiency syndrome (AIDS). These natural hosts maintain high SIV viral loads, but avoid immunodeficiency. Elucidating the mechanisms that allow natural hosts to coexist with SIV without overt disease may provide crucial information for understanding AIDS pathogenesis. Over the past few years, several key features of natural SIV infections have been described in studies conducted predominantly in sooty mangabeys (SMs), African green monkeys (AGMs), and mandrills. Natural SIV hosts are able to avoid the chronic, generalized immune system activation that is associated with disease progression in HIV-infected individuals and are known to downmodulate the expression of the receptors for SIV. In this perspective we propose that a critical factor that differentiates nonprogressive from progressive HIV or SIV infection is the maintenance of T cell immune competence in the face of a virus that infects and kills CD4(+) T cells. Elucidation of the mechanisms underlying the preservation of immune function during and after the acute phase of natural SIV infection may lead to the design of novel preventive and therapeutic interventions for treatment of chronic HIV infection.


[Technic and results of ureteroscopy for ureteral lithiasis. Apropos of 54 ureteroscopies].
Rigid ureteroscopy has been performed 54 times in 49 patients with ureteral stones. In 72% of the cases stones were removed or disintegrated with the ultrasound or electrohydraulic probe. Best results were obtained in the pelvic ureter (96,5% success). In the lumbar ureter only 56% success was obtained. Complications were observed in 15% of the cases, however only one complication necessitated an immediate open surgical operation.


A congenital malformation of the maxilla of a horse.
A 1-month-old male American Paint Horse was evaluated for a hard swelling on the right side of the maxillary region. On radiographs there was a large, expansile outpouching of the right maxillary bone between the second and third premolar teeth. Computed tomography further characterized the expansile lesion to have a soft tissue component and to originate in the region of a caudal maxillary tooth. Surgical reconstruction of the defect was unsuccessful and the animal was euthanetized. Based on failure to find histopathologic evidence of a neoplasm or cyst, the diagnosis was a congenital malformation.


Structural insights into the agonists binding and receptor selectivity of human histamine H4 receptor.
Histamine is a biogenic amine that participates in allergic and inflammatory processes by stimulating histamine receptors. The histamine H4 receptor (H4R) is a potential therapeutic target for chronic inflammatory diseases such as asthma and atopic dermatitis. Here, we show the cryo-electron microscopy structures of the H4R-Gq complex bound with an endogenous agonist histamine or the selective agonist imetit bound in the orthosteric binding pocket. The structures demonstrate binding mode of histamine agonists and that the subtype-selective agonist binding causes conformational changes in Phe3447.39, which, in turn, form the "aromatic slot". The results provide insights into the molecular underpinnings of the agonism of H4R and subtype selectivity of histamine receptors, and show that the H4R structures may be valuable in rational drug design of drugs targeting the H4R.


Simulation of oxygen transport and estimation of tissue perfusion in extensive microvascular networks: Application to cerebral cortex.
Advanced imaging techniques have made available extensive three-dimensional microvascular network structures. Simulation of oxygen transport by such networks requires information on blood flow rates and oxygen levels in vessels crossing boundaries of the imaged region, which is difficult to obtain experimentally. Here, a computational method is presented for estimating blood flow rates, oxygen levels, tissue perfusion and oxygen extraction, based on incomplete boundary conditions. Flow rates in all segments are estimated using a previously published method. Vessels crossing the region boundary are classified as arterioles, capillaries or venules. Oxygen levels in inflowing capillaries are assigned based on values in outflowing capillaries, and similarly for venules. Convective and diffusive oxygen transport is simulated. Contributions of each vessel to perfusion are computed in proportion to the decline in oxygen concentration along that vessel. For a vascular network in the mouse cerebral cortex, predicted tissue oxygen levels show a broad distribution, with 99% of tissue in the range of 20 to 80 mmHg under reference conditions, and steep gradients near arterioles. Perfusion and extraction estimates are consistent with experimental values. A 30% reduction in perfusion or a 30% increase in oxygen demand, relative to reference levels, is predicted to result in tissue hypoxia.


Side effects of X-ray irradiation on flight ability of Cydia pomonella moth.
The sterile insect technique (SIT) has proven to be an effective approach in managing the population of major invasive pests Our previous studies showed that irradiation of males at a dosage of 366 Gy X-rays resulted in complete sterility. However, the mating competitiveness of sterilized males is significantly compromised, which can be attributed to a decline in their ability to fly. In this study, we examined the flight patterns of both male and female adults of C. pomonella. The results revealed significant variations in the average flight speed of both genders at different stages of maturity, with females displaying longer flight duration and covering greater distances. Effect of irradiation on the flight performance of 3-day-old male moths was further evaluated, as they demonstrated the longest flight distance. The findings indicated a significant decrease in flight distance, duration, and average speed, due to wing deformities caused by irradiation, which also limited the dispersal distance of moths in orchards, as indicated by the mark-and-recapture assay. Quantitative reverse-transcription PCR analysis revealed a down-regulation of flight-related genes such as flightin, myosin heavy chain, and distal-less following radiation exposure. These findings demonstrate that X-ray irradiation at a radiation dose of 366 Gy has a detrimental effect on the flight ability of male C. pomonella adults. These insights not only contribute to a better understanding of how radiation sterilization diminishes the mating competitiveness of male moths, but also aid in the development and improvement of SIT practices for the effectively controlling C. pomonella. This article is protected by copyright. All rights reserved.


Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function.
Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.


Assessing adrenal insufficiency of corticosteroid secretion using free versus total cortisol levels in critical illness.
To study the value of free versus total cortisol levels in assessing relative adrenal insufficiency during critical illness-related corticosteroid insufficiency. A prospective study in a mixed intensive care unit from 2004 to 2007. We consecutively included 49 septic and 63 non-septic patients with treatment-insensitive hypotension in whom an adrenocorticotropic hormone (ACTH) test (250 μg) was performed. Serum total and free cortisol (equilibrium dialysis), corticosteroid-binding globulin (CBG) and albumin were assessed. Although a low CBG resulted in a high free cortisol level relative to total cortisol, free and total cortisol and their increases were well correlated (r = 0.77-0.79, P < 0.001). In sepsis, hypoalbuminemia did not affect total and free cortisol, and increases in total cortisol upon ACTH predicted increases in free cortisol regardless of low binding proteins. In non-sepsis, total cortisol was lower with than without hypoalbuminemia; free cortisol did not differ, since hypoalbuminemia concurred with a low CBG. Increases in total cortisol depended less on binding proteins than on raw levels. The areas under the receiver operating characteristic curve for predicting increases in free from total cortisol were 0.93-0.97 in sepsis and 0.79-0.85 in non-sepsis (P = 0.044 or lower for sepsis vs. non-sepsis). Although the biologically active free cortisol fraction depends on binding proteins, total cortisol correlates to free cortisol in treatment-insensitive hypotension during critical illness. In sepsis, albumin is not an important binding molecule. Subnormal increments in total cortisol upon ACTH suffice in assessing relative adrenal insufficiency, particularly in sepsis.


Men, trans/masculine, and non-binary people's experiences of pregnancy loss: an international qualitative study.
Growing numbers of men, trans/masculine, and non-binary people are becoming gestational parents, yet very little is known about experiences of pregnancy loss among this diverse population. The study employed a cross sectional design. Interviews were undertaken with a convenience sample of 51 trans/masculine and non-binary people who had undertaken at least one pregnancy, living in either Australia, the United States, Canada, or the European Union (including the United Kingdom). Participants were recruited by posts on Facebook and Twitter, via researcher networks, and by community members. 16 (31.2%) of the participants had experienced a pregnancy loss and are the focus of this paper. Thematic analysis was used to analyse interview responses given by these 16 participants to a specific question asking about becoming pregnant and a follow up probe question about pregnancy loss. Thematic analysis of interview responses given by the 16 participants led to the development of 10 themes: (1) pregnancy losses count as children, (2) minimizing pregnancy loss, (3) accounting for causes of pregnancy loss, (4) pregnancy loss as devastating, (5) pregnancy loss as having positive meaning, (6) fears arising from a pregnancy loss, (7) experiences of hospitals enacting inclusion, (8) lack of formal support offered, (9) lack of understanding from family, and (10) importance of friends. The paper concludes by outlining specific recommendations for clinical practice. These include the importance of focusing on the emotions attached to pregnancy loss, the need for targeted support services for men, trans/masculine, and non-binary people who undertake a pregnancy (including for their partners), and the need for ongoing training for hospital staff so as to ensure the provision of trans-affirming medical care.


Biostability of polyether-urethane scaffolds: a comparison of two novel processing methods and the effect of higher gamma-irradiation dose.
This article deals with enzyme-induced biodegradation behavior of thermoplastic polyether-urethane (TPU). Porous scaffolds were processed by a new foaming method applied in hot pressing and injection molding. The scaffolds were subsequently gamma sterilized. The samples were incubated with cholesterol esterase (CE) for 28 days to simulate an enzymatic degradation order to assess polymer biostability. The main focus of degradation products was the most toxic one: methylene dianiline (MDA). LC/MS was used to separate the breakdown products and to identify possible MDA amounts. The results showed that (a) the hot-pressed sample released an MDA amount almost twice as large (0.26 ng +/- 0.008) as that of the injection-molded samples (0.15 ng +/- 0.003) after incubation with enzyme activity in the physiological range, and (b) a tenfold increase in CE activity revealed considerably higher MDA amounts (7540.0 ng +/- 0.004). This enzyme concentration is physiologically unlikely, however, but may occur for extreme high inflammation behavior. Even for extremely high levels of CE enzyme, the scaffold will not discharge MDA above toxic levels. The injection-molded samples sterilized at 25 kGy seem to represent the most promising processing method. Therefore, the new injection-molding foaming process of polyether-urethane can be considered appropriate for use as a biomaterial.


Cannabinoid receptor 1 gene association with nicotine dependence.
The endogenous cannabinoid system has been implicated in drug addiction in animal models. The cannabinoid receptor 1 (CNR1) gene is 1 of the 2 receptors expressed in the brain. It has been reported to be associated with alcoholism and multiple drug abuse and dependence. To test the hypothesis that the CNR1 gene is associated with nicotine dependence. Genotype-phenotype association study. Ten single-nucleotide polymorphisms were genotyped in the CNR1 gene in 2 independent samples. For the first sample (n = 688), a 3-group case-control design was used to test allele association with smoking initiation and nicotine dependence. For the second sample (n = 961), association was assessed with scores from the Fagerström Test for Nicotine Dependence (FTND). Settings Population samples selected from the Mid-Atlantic Twin Registry. White patients aged 18 to 65 years who met the criteria of inclusion. Fagerström Tolerance Questionnaire and FTND scores. Significant single-marker and haplotype associations were found in both samples, and the associations were female specific. Haplotype 1-1-2 of markers rs2023239-rs12720071-rs806368 was associated with nicotine dependence and FTND score in the 2 samples (P < .001 and P = .009, respectively). Variants and haplotypes in the CNR1 gene may alter the risk for nicotine dependence, and the associations are likely sex specific.