[Mediolateral gradient of the nucleus accumbens nitrergic activation during exploratory behavior].
In Sprague-Dawley rats, by means of in vivo microdialysis combined with HPLC analysis it has been shown that an exploratory behavior in a new environment is accompanied by a rise in extracellular levels of citrulline (an NO co-product) in the mediolateral regions of the n. accumbens with the maximum observed in the medial n. accumbens. Infusions of 7-nitroindazole (0.5 mM), a neuronal NO synthase inhibitor, into the medial n. accumbens prevented the exploration-induced rise of extracellular citrulline levels in this area. The second presentation of the same chamber did not produce any significant changes of extracellular citrulline levels in the medial n. accumbens, although there was a tendency of a small increase. The presentation of a familiar chamber did not affect citrulline extracellular levels in this area. The data obtained indicate for the first time that exploratory activity in a new environment is accompanied by the nitrergic activation in the entire n. accumbens with the maximal activation in the medial part of this brain area.


Single-molecule analysis of genome rearrangements in cancer.
Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome.


Glucose dialysate induces mitochondrial DNA damage in peritoneal mesothelial cells.
It is known that peritoneal mesothelial cells (PMCs) are denuded in patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD); the mechanism of damage is not well known. A high quantity of glucose loaded onto PMCs in these patients may generate toxic radicals during the mitochondrial metabolism, leading to mitochondrial DNA damage that accumulates due to the incomplete repair system of this DNA. To study damage to the PMCs of long-term CAPD patients, and to examine whether glucose overload accelerates this damage in vitro. Descriptive clinical and in vitro study. Stable CAPD patients and nonuremic patients undergoing elective abdominal surgery. (1) Clinical Samples: 13 peritoneal tissue samples from CAPD patients and 5 omental tissue samples from patients with normal renal function were investigated. PMCs in dialysate effluent were collected from another 13 stable CAPD patients. (2) In Vitro Samples: Primary PMCs were incubated for up to 144 hours in medium containing one of the following: 5.6 mmol/L glucose (control), 56 mmol/L glucose (G), 222 mmol/L glucose (high G), or 222 mmol/L mannitol (high M; osmolar control for high G). The tissues and cells of clinical and in vitro samples were stained for light and immunoelectron microscopy with anti-8-hydroxy-2'-deoxyguanosine (anti-8-OH-dG) antibody, a marker of oxidative DNA damage. In vitro cells were also studied using transmission electron microscopy. Cellular ATP content, mitochondrial membrane potential, and intracellular generation of reactive oxygen species (ROS) were analyzed by luciferase-luciferin system, or by flow cytometry using rhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Biopsy specimens showed strong cytoplasmic staining with 8-OH-dG in patients on long-term CAPD, but only faint staining in patients with end-stage renal disease before the initiation of CAPD, and no staining in patients with normal renal function. Dialysate effluent showed strong granular staining with 8-OH-dG in most PMCs in all long-term CAPD patients, but only faint and focal staining in patients at the start and after 3-5 months of CAPD. In vitro experiments also showed strong granular staining by 8-OH-dG in most PMCs cultured in high G, weak staining in G and high M, and no staining in the control. Immunoelectron microscopy revealed the localization of 8-OH-dG to mitochondria. Transmission electron microscopy showed swelling of mitochondria, with decreased cristae, in PMCs cultured in high G. However, only partial expansion of mitochondria was seen in G and high M, and no changes were seen in the control. Cellular ATP content and mitochondrial membrane potential were reduced early, followed by an increase when cultured in high G. Intracellular ROS production was also increased in PMCs cultured in high G and high M. These data suggest that high-glucose peritoneal dialysate may promote oxidative mitochondrial DNA damage in PMCs in CAPD patients.


Self-assembly and adsorption of cetyltrimethylammonium bromide and didodecyldimethylammonium bromide surfactants at the mica-water interface.
The self-assembly and adsorption of the surfactants cetyltrimethylammonium bromide (CTAB) and didodecyldimethylammonium bromide (DDAB) at the muscovite mica-water interface are studied using molecular-dynamics simulations. Adsorption takes place by an ion-exchange mechanism, in which K+ ions are replaced by the organic alkylammonium cations from the solution. Simulations are performed with and without the surface K+ ions, with pure water, and with the surfactants in aqueous solution. CTAB and DDAB form micellar structures in bulk solution, and in the absence of the surface K+ ions, they quickly adsorb and form bilayer structures. The bilayer ordering of CTAB is not perfect, and there is a competition with the formation of cylindrical micelles. DDAB, on the other hand, forms a well-ordered bilayer structure, with the innermost layer showing strong orientational ordering, and the outermost layer being more disordered. The simulations with pure water highlight the molecular ordering and strong electrostatic interactions with the mica-surface atoms. Using simulated scattering length density profiles, the results are compared directly and critically with existing neutron reflectivity measurements. The simulation results are generally consistent with experiments, and yield new insights on the molecular-scale ordering at the mica-water interface.


Tissue-Specific Transcriptomics Reveals an Important Role of the Unfolded Protein Response in Maintaining Fertility upon Heat Stress in Arabidopsis.
High temperatures have a great impact on plant reproductive development and subsequent fruit and seed set, but the underlying molecular mechanisms are not well understood. We used transcriptome profiling to investigate the effect of heat stress on reproductive development of Arabidopsis thaliana plants and observed distinct response patterns in vegetative versus reproductive tissues. Exposure to heat stress affected reproductive developmental programs, including early phases of anther/ovule development and meiosis. Also, genes participating in the unfolded protein response (UPR) were enriched in the reproductive tissue-specific genes that were upregulated by heat. Moreover, we found that the UPR-deficient bzip28 bzip60 double mutant was sensitive to heat stresses and had reduced silique length and fertility. Comparison of heat-responsive wild type versus bzip28 bzip60 plants identified 521 genes that were regulated by bZIP28 and bZIP60 upon heat stress during reproductive stages, most of which were noncanonical UPR genes. Chromatin immunoprecipitation coupled with high-throughput sequencing analyses revealed 133 likely direct targets of bZIP28 in Arabidopsis seedlings subjected to heat stress, including 27 genes that were also upregulated by heat during reproductive development. Our results provide important insights into heat responsiveness in Arabidopsis reproductive tissues and demonstrate the protective roles of the UPR for maintaining fertility upon heat stress.


Robotic Telecytology for Remote Cytologic Evaluation without an On-site Cytotechnologist or Cytopathologist: An Active Quality Assessment and Experience of Over 400 Cases.
The first satellite center to offer interventional radiology procedures at Memorial Sloan Kettering Cancer Center opened in October 2014. Two of the procedures offered, fine needle aspirations and core biopsies, required a rapid on-site cytologic evaluation of smears and biopsy touch imprints for cellular content and adequacy. The volume and frequency of such evaluations did not justify hiring on-site cytotechnologists, and therefore, a dynamic robotic telecytology (TC) solution was created. In this article, we provide data on our experience with this active implementation. Sakura VisionTek was selected as our robotic TC solution. A retrospective analysis of all TC evaluations from this satellite site was performed. Information was collected on demographics, lesion location, imaging modality; a comparison of TC-assisted adequacy with final adequacy was also conducted. An analysis of 439 cases was performed over a period of 23 months with perfect correlation in 92.7% (407/439) of the cases. An adequacy upgrade (inadequate specimen becomes adequate) in 6.6% (29/439) of the cases. An adequacy downgrade (adequate specimen becomes inadequate), is near zero at 0.7% (3/439) of the cases. Dynamic robotic TC is effective for immediate evaluations performed without on-site cytotechnology staff. The overall intent of this article is to present data and concordance rates as outcome metrics. Thus far, such outcome metrics have exceeded our expectations. Our TC implementation shows high, perfect concordance. Adequacy upgrades are minor but more relevant and impressive is a near zero adequacy downgrade. Our full implementation has been so successful that plans are in place for configurations at future satellite sites.


Sensorimotor control in Parkinsonism.
Performance on two sensorimotor tasks was compared between 28 patients with Parkinsonism and 28 age and sex matched controls. One task involved complex motor activity (the Bead and Tapper test) and the other simple motor activity with a verbal component (the Letter Cancellation test). Both tests had three levels of complexity. Under conditions of increasing complexity in the performance required, the scores for the Parkinsonism subjects decreased disproportionately compared with the controls. This finding was more clearly established for the Bead and Tapper test than for the Letter Cancellation test. These results confirmed previous studies, and the nature of a possible central programming deficit was discussed. Laterality of brain involvement in the Parkinsonism subjects had no significant effect.


[Consequences of interactions between thymic lymphoid and epithelial cells in vitro].
A 24-hour co-cultivation of thymocytes and epithelial cells taken from human thymus results in mutual activation of epitheliocytes and thymocytes, as well as in apoptosis of thymocytes. The apoptosis can also be induced by a cultural supernatant of the thymic-epithelial cells, its level being lower, however, than in the co-culture. Thymocyte death and elimination develop faster in a co-culture with allogeneic thymic epithelial cells.


Evaluation of orthostatic hypotension: relationship of a new self-report instrument to laboratory-based measures.
To compare measured autonomic deficits (composite autonomic severity score [CASS]) with a brief self-report scale we developed to measure severity of symptoms of orthostatic hypotension. Patients were recruited in 2 phases: from August to October 2002 and in April 2004. All patients underwent full evaluation in the autonomic laboratory, from which a CASS of autonomic deficits was derived. Patients also completed the 5-item self-report Orthostatic Grading Scale, which inquires about symptoms of orthostatic intolerance due to orthostatic hypotension (eg, severity, frequency, and interference with daily activities). Of 145 patients, 97 (67%) had orthostatic hypotension. The 5-item scale demonstrated strong internal consistency (coefficient alpha=.91). Patients with orthostatic hypotension had significantly higher scores on each questionnaire item and CASS subscores than those without orthostatic hypotension. The scale items correlated significantly with each of the CASS subscores, maximally with the CASS adrenergic subscore. Orthostatic hypotension is not the only cause of reduced orthostatic tolerance, and some patients may have orthostatic hypotension but be asymptomatic. Results of this study indicate that this 5-item questionnaire is a reliable and valid measure of the severity of symptoms of orthostatic hypotension and that it can supplement laboratory-based measures to provide a rapid, more complete clinical assessment. This questionnaire would also be useful as a brief screening device for orthostatic intolerance to aid physicians in identifying patients who may have orthostatic hypotension.


Construction of 2D layered TiO2@MoS2heterostructure for efficient adsorption and photodegradation of organic dyes.
In this work, heterostructures of coupled TiO2@MoS2with different phases of MoS2were synthesized via hydrothermal technique. The prepared materials were thoroughly characterized using various techniques, including XRD, SEM, transmission electron microscopy, Brunauer-Emmet-Teller, XPS, Zeta potential and UV-vis spectroscopy. The optimized nanocomposites were tested for the photocatalytic degradation of methyl Orange (MO) under visible light as well as the adsorption of Rhodamine b (RhB) and methelene blue (MB) dyes. The TiO2@1T/2H-MoS2heterostructures exhibited a narrow bandgap compared to the other studied nanomaterials. A remarkable photodegradation efficiency of TiO2@1T/2H-MoS2was observed, which completely degraded 20 ppm of MO after 60 min with high stability over four successive cycles. This can be assigned to the formation of unique heterostructures with aligned energy bands between MoS2nanosheets and TiO2nanobelts. The formation of these novel interfaces promoted the electron transfer and increased the separation efficiency of carriers, resulting in high photocatalytic degradation. Furthermore, the adsorption efficiency of TiO2@1T/2H-MoS2was unique, 20 ppm solutions of RhB and MB were removed after 1 and 2 min, respectively. The superior adsorption performance of the TiO2@1T/2H-MoS2can be attributed to its high surface area (279.9 m2g-1) and the rich concentration of active sites. The kinetics and the isothermal analysis revealed that the TiO2@1T/2H MoS2heterstructures have maximum adsorption capacity of 1200 and 970 mg g-1for RhB and MB, respectively. This study provides a powerful way for designing an effective photocatalyst and adsorbent TiO2-based nanocomposites for water remediation.


Toward Autonomous Pulmonary Artery Catheterization: A Learning-based Robotic Navigation System.
Providing imaging during interventional treatments of cardiovascular diseases is challenging. Magnetic Resonance Imaging (MRI) has gained popularity as it is radiation-free and returns high resolution of soft tissue. However, the clinician has limited access to the patient, e.g., to their femoral artery, within the MRI scanner to accurately guide and manipulate an MR-compatible catheter. At the same time, communication will need to be maintained with a clinician, located in a separate control room, to provide the most appropriate image to the screen inside the MRI room. Hence, there is scope to explore the feasibility of how autonomous catheterization robots could support the steering of catheters along trajectories inside complex vessel anatomies.In this paper, we present a Learning from Demonstration based Gaussian Mixture Model for a robot trajectory optimisation during pulmonary artery catheterization. The optimisation algorithm is integrated into a 2 Degree-of-Freedom MR-compatible interventional robot allowing for continuous and simultaneous translation and rotation. Our methodology achieves autonomous navigation of the catheter tip from the inferior vena cava, through the right atrium and the right ventricle into the pulmonary artery where an interventions is performed. Our results show that our MR-compatible robot can follow an advancement trajectory generated by our Learning from Demonstration algorithm. Looking at the overall duration of the intervention, it can be concluded that procedures performed by the robot (teleoperated or autonomously) required significantly less time compared to manual hand-held procedures.


Myofunctional therapy. Part 1: Culture, industrialisation and the shrinking human face.
Culture, industrialisation and the shrinking human face: Why is it important? Over the past 300,000 years, not only has the way we consume food from birth through our lifetime changed, there have also been changes related to the methods of food preparation, availability, processing, and storage. These diet-related factors, along with other epigenetic factors, have led to a widespread increase in orofacial myofunctional disorders (OMDs) and resultant human malocclusion phenotypes (HMPs) worldwide. Currently there is an increasing need for resolution of HMPs in early childhood and associated OMDs. This review will include reports of cases and describe the nature of the problem and strategies for effective solutions.


Utility values for specific chronic myeloid leukemia chronic phase health states from the general public in the United Kingdom.
This study elicited time trade-off (TTO) and standard gamble (SG) preference values associated with four health states corresponding to response levels in chronic phase chronic myeloid leukemia (CML) from members of the general public in the UK (n = 235). Health states studied were treatment-free remission (TFR), complete molecular response (CMR, i.e. undetectable disease on treatment), molecular response and reappearance of detectable disease (i.e. relapse from TFR to molecular response requiring treatment). TFR was the most preferred health state (mean utility of 0.97 [TTO] and 0.87 [SG]) followed by CMR (mean utility of 0.96 [TTO] and 0.85 [SG]) followed by molecular response (mean utility of 0.94 [TTO] and 0.80 [SG]) followed by reappearance of detectable disease (mean utility of 0.90 [TTO] and 0.72 [SG]). SG values were significantly lower than TTO values (p < 0.001). The study demonstrated that different treatment responses may impact on the health-related quality of life of patients with chronic phase CML.


Minimizing Precision-Weighted Sensory Prediction Errors via Memory Formation and Switching in Motor Adaptation.
Humans predict the sensory consequences of motor commands by learning internal models of the body and of environment perturbations. When facing a sensory prediction error, should we attribute this error to a change in our body, and update the body internal model, or to a change in the environment? In the latter case, should we update an existing perturbation model or create a new model? Here, we propose that a decision-making process compares the models' prediction errors, weighted by their precisions, to select and update either the body model or an existing perturbation model. When no model can predict a perturbation, a new perturbation model is created and selected. When a model is selected, both the prediction's mean estimate and uncertainty are updated to minimize future prediction errors and to increase the precision of the predictions. Results from computer simulations, which we verified in an arm visuomotor adaptation experiment with subjects of both sexes, account for short aftereffects and large savings after adaptation to large, but not small, perturbations. Results also clarify previous data in the absence of errors (error-clamp): motor memories show an initial lack of decay after a large perturbation, but gradual decay after a small perturbation. Finally, qualitative individual differences in adaptation were explained by subjects selecting and updating either the body model or a perturbation model. Our results suggest that motor adaptation belongs to a general class of learning according to which memories are created when no existing memories can predict sensory data accurately and precisely.SIGNIFICANCE STATEMENT When movements are followed by unexpected outcomes, such as following the introduction of a visuomotor or a force field perturbation, or the sudden removal of such perturbations, it is unclear whether the CNS updates existing memories or creates new memories. Here, we propose a novel model of adaptation, and investigate, via computer simulations and behavioral experiments, how the amplitude and schedule of the perturbation, as well as the characteristics of the learner, lead to the selection and update of existing memories or the creation of new memories. Our results provide insights into a number of puzzling and contradictory motor adaptation data, as well as into qualitative individual differences in adaptation.


Retroviral infection is limited by Brownian motion.
Replication-defective retroviruses are frequently used as gene carriers for gene transfer into target cells. Here we show that the short half-lives of retroviruses limit the distance that they can effectively travel in solution by Brownian motion, and thus the possibility of successful gene transfer. This physiochemical limitation can be overcome, and effective contact between the retroviral gene carrier and the target cell can be obtained, by using net convective flow of retrovirus-containing medium through a layer of target cells. Using model cell lines (NIH-3T3 and CV-1), it was shown that gene transfer rates can be increased by more than an order of magnitude using the same concentration infection medium. High transduction rates could be obtained even in the absence of polycations, such as Polybrene, which heretofore have been required to achieve reasonable transduction rates. This development may play an important role in realizing human gene therapy.


Ankle synovectomy: an approach to the rheumatoid ankle.
Rheumatoid synovitis or pannus formation is chiefly responsible for pain, joint destruction, and eventual erosion in patients afflicted with rheumatoid arthritis. Synovectomy is a nondestructive means of arresting this inflammatory process in the rheumatoid ankle. The technique of ankle synovectomy is described.


Prone positioning in acute respiratory distress syndrome (ARDS): when and how?
Acute respiratory distress syndrome (ARDS) is a severe form of respiratory failure. It remains one of the most devastating conditions in the intensive care unit. Mechanical ventilation with positive end-expiratory pressure is a cornerstone therapy for ARDS patients. One adjuvant alternative is to place the patient in a prone position. Since it was first described in 1976, prone positioning has been safely employed to improve oxygenation in many patients with ARDS. Prone positioning may also minimize secondary lung injury induced by mechanical ventilation, although this benefit has not been investigated as extensively, despite its potential. In spite of a strong physiological justification, prone positioning is still not widely accepted as an adjunct therapy in ARDS patients and it is only used regularly in only 10% of ICUs. This may be explained in part by the reluctance to change position, risks and unclear effects on relevant outcomes. In this paper, we review all aspects of prone positioning, from the pathophysiology to the clinical studies of patient outcome, and we also discuss the latest controversies surrounding this treatment.


[Risk factors for aerostasis failure in case of lung resection].
The results of risk factors analysis of aerostasis failure in 141 patients are presented in the article. All patients were operated in the A.V. Vishnevskogo Institute of Surgery at the period from January 2009 to December 2010. During statistical analysis it was considered surgeries volume, presence of emphysema, respiratory function etc. Also it was defined the dependence of venting duration in patients with residual pleural cavity and long aerostasis failure.


Memory in autism: review and synthesis.
Much research about memory in autism concerns the hypothesis that autism is similar to adult-onset amnesia. Initial support for the hypothesis came from post-mortem studies of individuals with autism showing abnormalities in the hippocampus and related brain structures, as well as behavioral studies finding contrasts between intact cued recall and impaired free recall and recognition in autism. The hypothesis was later brought into question by the finding of intact performance in individuals with autism on explicit memory tasks typically impaired in adult-onset amnesia. The present paper proposes a possible reconciliation of these contradictory findings, suggesting that there is selective damage to the limbic-prefrontal episodic memory system, sparing the limbic-only perceptual representation system, and the semantic memory system. This view is consistent with other evidence for early selective damage to other systems involving cooperation between the limbic system and the medial prefrontal cortex in autism.


Assessment of the Association between In Vivo Corneal Morphogeometrical Changes and Keratoconus Eyes with Severe Visual Limitation.
Assessing changes suffered by the cornea as keratoconus progresses has proven to be vital for this disease diagnosis and treatment. This study determines the corneal biometric profile in eyes considered as affected by keratoconus (KC) showing severe visual limitation, by means of in vivo 3D modelling techniques. This observational case series study evaluated new objective indices in 50 healthy and 30 KC corneas, following a validated protocol created by our research group, which has been previously used for diagnosis and characterization of KC in asymptomatic (preclinical) and mild visually impaired eyes. Results show a statistically significant reduction of corneal volume and an increase of total corneal area in the severe KC group, being anterior and posterior corneal surfaces minimum thickness points the best correlated parameters, although with no discrimination between groups. Receiving operator curves were used to determine sensitivity and specificity of selected indices, being anterior and posterior apex deviations the ones which reached the highest area under the curve, both with very high sensitivity (96.7% and 90%, respectively) and specificity (94.0% and 99.9%, respectively). The results suggest that once severe visual loss appears, anterior corneal topography should be considered for a more accurate diagnosis of clinical KC, being anterior apex deviation the key metric discriminant. This study can be a useful tool for KC classification, helping doctors in diagnosing severe cases of the disease, and can help to characterize corneal changes that appear when severe KC is developed and how they relate with vision deterioration.


A general and efficient cantilever functionalization technique for AFM molecular recognition studies.
Atomic force microscopy (AFM) is a versatile technique for the investigation of noncovalent molecular associations between ligand-substrate pairs. Surface modification of silicon nitride AFM cantilevers is most commonly achieved using organic trialkoxysilanes. However, susceptibility of the Si−O bond to hydrolysis and formation of polymeric aggregates diminishes attractiveness of this method for AFM studies. Attachment techniques that facilitate immobilization of a wide variety of organic and biological molecules via the stable Si−C bond on silicon nitride cantilevers would be of great value to the field of molecular recognition force spectroscopy. Here, we report (1) the formation of stable, highly oriented monolayers on the tip of silicon nitride cantilevers and (2) demonstrate their utility in the investigation of noncovalent protein-ligand interactions using molecular recognition force spectroscopy. The monolayers are formed through hydrosilylation of hydrogen-terminated silicon nitride AFM probes using a protected α-amino-ω-alkene. This approach facilitates the subsequent conjugation of biomolecules. The resulting biomolecules are bound to the tip by a strong Si−C bond, completely uniform with regard to both epitope density and substrate orientation, and highly suitable for force microscopy studies. We show that this attachment technique can be used to measure the unbinding profiles of tip-immobilized lactose and surface-immobilized galectin-3. Overall, the proposed technique is general, operationally simple, and can be expanded to anchor a wide variety of epitopes to a silicon nitride cantilever using a stable Si−C bond.


Mesomorphism of a new series of catanionic 4-(1-pentylheptyl)benzenesulfonates.
The effect of cationic chemical structure on the mesophase behavior, and self-assembly of catanionic compounds is studied through a systematic structure variation of six cationic surfactants with anionic sodium 4-(1-pentylheptyl)benzenesulfonate surfactant. A series of cationic surfactants are selected with an increasing number of n-dodecyl chains on the same ammonium headgroup (from one to three), and with an increasing number of ammonium headgroups (from two to four). Thermal and phase behavior of the synthesized products were examined by thermogravimetry, polarizing microscopy, differential scanning calorimetry, powder and grazing incidence X-ray diffraction, and molecular simulation. The compounds are complexes of high thermal stability. Most of the thermotropic mesophases are smectic, although the hexagonal mesophase is also observed. Introduction of the third dodecyl chain into the cationic part leads to pronounced lower melting temperatures and eradicates mesomorphism, while the addition of a fourth ammonium headgroup leads to a catanionic benzenesulfonate that decomposes simultaneously under melting. Melting and crystallization temperatures show nonlinear dependence on the total number of alkyl chains, and on the number of headgroups in the molecule. Molecular dynamics simulation of the smectic phase points to separated hydrophilic and hydrophobic layers, favoring head-to-head packing of antiparallel molecules. On the other hand, there are two ion pairs in the column cross-section of the hexagonal LC phase of didodecyldimethylammonium-4-(1-pentylheptyl)benzenesulfonate.


Developing Effective and Efficient care pathways in chronic Pain: DEEP study protocol.
Pain affecting the face or mouth and lasting longer than three months ("chronic orofacial pain", COFP) is relatively common in the UK. This study aims to describe and model current care pathways for COFP patients, identify areas where current pathways could be modified, and model whether these changes would improve outcomes for patients and use resources more efficiently. The study takes a prospective operations research approach. A cohort of primary and secondary care COFP patients (n = 240) will be recruited at differing stages of their care in order to follow and analyse their journey through care. The cohort will be followed for two years with data collected at baseline 6, 12, 18, and 24 months on: 1) experiences of the care pathway and its impacts; 2) quality of life; 3) pain; 4) use of health services and costs incurred; 5) illness perceptions. Qualitative in-depth interviews will be used to collect data on patient experiences from a purposive sub-sample of the total cohort (n = 30) at baseline, 12 and 24 months. Four separate appraisal groups (public, patient, clincian, service manager/commissioning) will then be given data from the pathway analysis and asked to determine their priority areas for change. The proposals from appraisal groups will inform an economic modelling exercise. Findings from the economic modelling will be presented as incremental costs, Quality Adjusted Life Years (QALYs), and the incremental cost per QALY gained. At the end of the modelling a series of recommendations for service change will be available for implementation or further trial if necessary. The recent white paper on health and the report from the NHS Forum identified chronic conditions as priority areas and whilst technology can improve outcomes, so can simple, appropriate and well-defined clinical care pathways. Understanding the opportunity cost related to care pathways benefits the wider NHS. This research develops a method to help design efficient systems built around one condition (COFP), but the principles should be applicable to a wide range of other chronic and long-term conditions.


Use of FK506 immunosuppressive therapy in pancreas transplantation.
The purpose of this study was to evaluate the safety, efficacy, and transplant outcomes associated with FK506 rescue and maintenance therapy in pancreas transplant recipients. A chart review was conducted on 10 patients receiving FK506 after pancreas transplantation. Transplant outcomes were compared with an equivalent group of patients receiving cyclosporine. Medication dose, side effects, infections, rejection episodes, glycemic control, and graft survival were recorded from 2 to 28 weeks after transplant. Rescue therapy was successful in the patients who were converted to FK506 prior to a significant decline in glycemic control, whereas those patients who were converted after a decline in glycemic control were required to return to exogenous insulin administration. Neurological complications, nephrotoxicity, incidence of infection, hypertension, rejection, and graft survival were similar for both groups. Use of FK506 is comparable to cyclosporine in pancreas allograft recipients and successful conversion from cyclosporine to FK506 can be undertaken for rescue therapy.


Core crystallization and pile-up in the cooling sequence of evolving white dwarfs.
White dwarfs are stellar embers depleted of nuclear energy sources that cool over billions of years1. These stars, which are supported by electron degeneracy pressure, reach densities of 107 grams per cubic centimetre in their cores2. It has been predicted that a first-order phase transition occurs during white-dwarf cooling, leading to the crystallization of the non-degenerate carbon and oxygen ions in the core, which releases a considerable amount of latent heat and delays the cooling process by about one billion years3. However, no direct observational evidence of this effect has been reported so far. Here we report the presence of a pile-up in the cooling sequence of evolving white dwarfs within 100 parsecs of the Sun, determined using photometry and parallax data from the Gaia satellite4. Using modelling, we infer that this pile-up arises from the release of latent heat as the cores of the white dwarfs crystallize. In addition to the release of latent heat, we find strong evidence that cooling is further slowed by the liberation of gravitational energy from element sedimentation in the crystallizing cores5-7. Our results describe the energy released by crystallization in strongly coupled Coulomb plasmas8,9, and the measured cooling delays could help to improve the accuracy of methods used to determine the age of stellar populations from white dwarfs10.


miRNA-ome plasma analysis unveils changes in blood-brain barrier integrity associated with acute liver failure in rats.
Hepatic encephalopathy (HE) symptoms associated with liver insufficiency are linked to the neurotoxic effects of ammonia and other toxic metabolites reaching the brain via the blood-brain barrier (BBB), further aggravated by the inflammatory response. Cumulative evidence documents that the non-coding single-stranded RNAs, micro RNAs (miRs) control the BBB functioning. However, miRs' involvement in BBB breakdown in HE is still underexplored. Here, we hypothesized that in rats with acute liver failure (ALF) or rats subjected to hyperammonemia, altered circulating miRs affect BBB composing proteins. Transmission electron microscopy was employed to delineate structural alterations of the BBB in rats with ALF (thioacetamide (TAA) intraperitoneal (ip.) administration) or hyperammonemia (ammonium acetate (OA) ip. administration). The BBB permeability was determined with Evans blue dye and sodium fluorescein assay. Plasma MiRs were profiled by Next Generation Sequencing (NGS), followed by in silico analysis. Selected miRs, verified by qRT-PCR, were examined in cultured rat brain endothelial cells. Targeted protein alterations were elucidated with immunofluorescence, western blotting, and, after selected miR mimics transfection, through an in vitro resistance measurement. Changes in BBB structure and increased permeability were observed in the prefrontal cortex of TAA rats but not in the brains of OA rats. The NGS results revealed divergently changed miRNA-ome in the plasma of both rat models. The in silico analysis led to the selection of miR-122-5p and miR-183-5p with their target genes occludin and integrin β1, respectively, as potential contributors to BBB alterations. Both proteins were reduced in isolated brain vessels and cortical homogenates in TAA rats. We documented in cultured primary brain endothelial cells that ammonia alone and, in combination with TNFα increases the relative expression of NGS-selected miRs with a less pronounced effect of TNFα when added alone. The in vitro study also confirmed miR-122-5p-dependent decrease in occludin and miR-183-5p-related reduction in integrin β1 expression. This work identified, to our knowledge for the first time, potential functional links between alterations in miRs residing in brain endothelium and BBB dysfunction in ALF.


Clinical features of influenza C virus infection in children.
Seroepidemiological studies have revealed that influenza C virus is widely distributed globally. However, because the isolation of this virus is difficult, there have been few reports on its clinical features. Between December 1990 and November 2004, 84,946 respiratory-tract specimens were obtained from patients < or = 15 years old. On the basis of the results of isolation of virus, we examined the clinical data on children infected with influenza C virus. Of 170 children infected with influenza C virus, 157 (92.4%) were < 6 years old. Fever (frequency, 90.0%), cough (frequency, 74.1%), and rhinorrhea (frequency, 61.8%) were the most frequent symptoms. The mean duration of fever was 2.88 days (standard deviation, 1.66 days). Of the 170 children, 29 were hospitalized, and 21 (72.4%) of these 29 had lower-respiratory-tract illness such as pneumonia, bronchitis, and bronchiolitis. The rate of hospital admission was significantly higher in children < 2 years old than in children 2-5 years old (30.4% vs. 11.9%; P = .0043). Influenza C virus is a significant cause of upper-respiratory-tract illness in children < 6 years old, and the risk of complications with lower-respiratory-tract illness is particularly high in children < 2 years old.


Arteriovenous shunt measurement during endovascular therapy for cerebrospinal lesions.
To determine (a) whether superselective angioscintigraphy with technetium-99m macroaggregated albumin (99mTc-MAA) can be used for the evaluation of arteriovenous shunting in tumors and vascular malformations of the head and spine and (b) whether the amount of microparticles shunted is related to diagnosis, lesion size, or angiographic pattern. Particles of 99mTc-MAA with a calibrated diameter of 25 to 50 microm were delivered intraarterially in feeders of head and spine tumors and vascular malformations in 38 patients. The first estimation of the proportion of particles reaching the lungs was made on-line in the angiography suite using a hand-held lead-shielded detector. Evaluation of the intralesional shunt (pulmonary shunt index, or PSI) was derived from quantitative gamma camera recordings of tumoral and pulmonary activity after the embolization procedure was complete. The PSI value ranged from 48% to 100% for vascular malformations and vascular tumors (n = 11), 82% to 95% for juvenile angiofibromas (n = 4), 63% to 70% for high-grade gliomas (n = 2), 0% to 50% for renal cell carcinoma metastases (n = 4), 0% to 86% for meningiomas (n = 11), and 0% to 36% for paragangliomas (n = 6). Angiographically, the presence of visible arteriovenous channels was predictive of a high PSI. In contrast, the presence of early venous drainage was associated with a wide PSI range. Superselective 99mTc-MAA angioscintigraphy of tumors and vascular malformations of the head and spine is a valuable method for quantifying an intralesional arteriovenous shunt before embolization.


[Characteristics of circadian variability of heart rate during Holter monitoring].
24-hour Holter monitoring of 60 healthy children aged 3 to 15 years provided criteria for assessing the circadian heart rate (HR) variations as a measure of alterations occurred in autonomic heart rhythm control. Specific HR dynamics during their sleep is described, which involves changes in steady-state rhythm and higher dispersion periods associated with the alternation of slow and fast sleep phases. Sex- and age-related HR features are defined during 24-hour monitoring.


Enhanced neural responses to self-triggered voice pitch feedback perturbations.
This study investigated the effect of self-triggered voice fundamental frequency (F0) feedback perturbation on auditory event-related potentials (ERPs) during vocalization and listening. Auditory ERPs were examined in response to self-triggered and computer-triggered -200 cents pitch-shift stimuli while participants vocalized or listened to the playback of their self-vocalizations. The stimuli were either presented with a delay of 500-1000 ms after the participants pressed a button or delivered by a computer with an interstimulus interval of 500-1000 ms. Results showed that self-triggered stimuli elicited larger ERPs compared with computer-triggered stimuli during both vocalization and listening conditions. These findings suggest that self-triggered perturbation of self-vocalization auditory feedback may enhance auditory responses to voice feedback pitch perturbation during vocalization and listening.


The association between A Body Shape Index and mortality: Results from an Australian cohort.
It is well recognised that obesity increases the risk of premature death. A Body Shape Index (ABSI) is a formula that uses waist circumference (WC), body mass index (BMI) and height to predict risk of premature mortality, where a high score (Quartile 4) indicates that a person's WC is more than expected given their height and weight. Our study examines the association between ABSI quartiles and all-cause-, cardiovascular- and cancer-related mortality, and primary cause of death. Self-reported demographic and biomedically measured health-related risk factor and weight data was from the baseline stage of the North West Adelaide Health Study (1999-2003, n = 4056), a longitudinal cohort of Australian adults. Death-related information was obtained from the National Death Index. Primary cause of death across ABSI quartiles was examined. The association between mortality and ABSI (quartile and continuous scores) was investigated using a Cox proportional hazards survival model and adjusting for socioeconomic, and self-reported and biomedical risk factors. The proportion of all three types of mortality steadily increased from ABSI Quartile 1 through to Quartile 4. After adjusting for demographic and health-related risk factors, the risk of all-cause mortality was higher for people in ABSI Quartile 4 (HR 2.64, 95% CI 01.56-4.47), and ABSI Quartile 3 (HR 1.95, 95% CI 1.15-3.33), with a moderate association for the continuous ABSI score (HR 1.32, 95% CI 1.18-1.48). ABSI is therefore positively associated with mortality in Australian adults. Different combined measures of obesity such as the ABSI are useful in examining mortality risk.


Portable chamber system for measuring chloroform fluxes from terrestrial environments--methodological challenges.
This study describes a system designed to measure chloroform flux from terrestrial systems, providing a reliable first assessment of the spatial variability of flux over an area. The study takes into account that the variability of ambient air concentrations is unknown. It includes quality assurance procedures, sensitivity assessments, and testing of materials used to ensure that the flux equation used to extrapolate from concentrations to fluxes is sound and that the system does not act as a sink or a source of chloroform. The results show that many materials and components commonly used in sampling systems designed for CO2, CH4, and N2O emit chloroform and other volatile chlorinated compounds (VOCls) and are thus unsuitable in systems designed for studies of such compounds. To handle the above-mentioned challenges, we designed a system with a non-steady-state chamber and a closed-loop air-circulation unit returning scrubbed air to the chamber. Based on empirical observations, the concentration increase during a deployment was assumed to be linear. Four samples were collected consecutively and a line was fitted to the measured concentrations. The slope of the fitted line and the y-axis intercept were input variables in the equation used to transform concentration change data to flux estimates. The soundness of the flux equation and the underlying assumptions were tested and found to be reliable by comparing modeled and measured concentrations. Fluxes of chloroform in a forest clear-cut on the east coast of Vancouver Island, BC, during the year were found to vary from -130 to 620 ng m(-2) h(-1). The study shows that the method can reliably detect differences of approximately 50 ng m(-2) h(-1) in chloroform fluxes. The statistical power of the method is still comparatively strong down to differences of 35 ng m(-2) h(-1), but for smaller differences, the results should be interpreted with caution.


Two rigor states in skinned crayfish single muscle fibers.
We studied the tension and stiffness of crayfish skinned single muscle fibers during and after the induction of rigor by removal of MgATP (substrate). We found that the rigor state is not unique but depends on the condition of the muscle before rigor. Fibers induced into rigor with a minimum of activation (low rigor) develop a small tension and moderate stiffness, while those entering rigor during maximum activation (high rigor) maintain near peak tension (80%) and develop a high stiffness. These rigor states are insensitive to Ca addition or deletion but they are partially interconvertible by length change. Stiffness changes when the rigor muscle length is varied, a condition in which the number of attached cross-rigor muscle length is varied, a condition in which the number of attached cross-bridges cannot change, and high-rigor muscle becomes less stiff than low-rigor muscle when the former is brought to the same tension by length release. The sensitivity of low, high, or length-released high-rigor muscles to trace substrate concentration (less than muM) differs, and rigor at lower strain is more suscepitible to substrate.


Comparison between European Medicines Agency and US Food and Drug Administration in Granting Accelerated Marketing Authorizations for Covid-19 Medicines and their Utilized Regulations.
Prompted by the Covid-19 pandemic and the need to ensure timely and safe access to medicines during a pandemic, the aim of this study was to compare and contrast the EU and US regulations, processes, and outcomes pertaining to the granting of accelerated Marketing Authorizations (MAs) for COVID-19 vaccines and treatments with a view to determining how effective these regulations were in delivering safe medicines in a timely manner. MAs for medicines approved for Covid-related indications in the first two pandemic years (March 2020-February 2022) were identified using the European Medicines Agency (EMA) and US Food and Drug Administration (FDA) websites. Authorization reports and utilized regulations were reviewed to determine and compare approval timelines, facilitated pathways, accepted clinical evidence, and effectiveness of the regulations by assessing them against time and safety standards. By the end of February 2022, the EMA and FDA had granted 12 and 14 MAs, respectively. Two EU and two US approvals were issued in relation to new indications for already-approved treatments; the remaining ones were first-time approvals of novel vaccines and treatments. The median time to approval was 24 days for the EMA's conditional MAs and 36 days for the USFDA's Emergency Use Authorizations (EUA) for all Covid-19 medicines. This is compared with 23 and 28 days, respectively, specifically for first-time novel vaccines and treatments authorized by both USFDA and EMA. The USFDA and EMA differed markedly in terms of the time taken to approve new indications of already-approved treatment; the USFDA took 65 days for such approval, compared with 133 days for the EMA. Where MAs were issued by both authorities, USFDA approvals were issued before EMA approvals; applications for approval were submitted to the FDA before submission to the EMA. Three EU and two US MAs were based on data from two or more phase 3 clinical trials; the remaining ones were based on single trial data. Only six EU and four US trials had been completed by the time of authorization. This was in line with regulations. While the applicable regulations shared many similarities, there were marked differences. For instance, the EU's conditional MA regulation pertains only to first approvals of new treatments. It does not cover new indications of already-approved treatments. This contrasts with the US, where the EUA regulation applies to both types of applications, something that may have impacted approval timelines. Overall, both EU and US utilized regulations were considered to be effective. For most cases, utilizing such regulations for Covid-19 MAs resulted in faster approval timelines compared to standard MAs. They were flexible enough to manage the process of granting emergency approvals while maintaining strict requirements and allowing comprehensive reviews of the supporting evidence. US and EU regulations were effective in ensuring timely accelerated market access to Covid-19 medicines during the pandemic without compromising the approval standards related to safety or efficacy. The population in both regions will receive comparable access to medicines during a pandemic if sponsors submit their applications to both authorities in parallel.


Low-Power, 8-Channel EEG Recorder and Seizure Detector ASIC for a Subdermal Implantable System.
EEG remains the mainstay test for the diagnosis and treatment of patients with epilepsy. Unfortunately, ambulatory EEG systems are far from ideal for patients who have infrequent seizures. These systems only last up to 3 days and if a seizure is not captured during the recordings, a definite diagnosis of the patient's condition cannot be given. This work aims to address this need by proposing a subdermal implantable, eight-channel EEG recorder and seizure detector that has two modes of operation: diagnosis and seizure counting. In the diagnosis mode, EEG is continuously recorded until a number of seizures are recorded. In the seizure counting mode, the system uses a low-power algorithm to track the number of seizures a patient has, providing doctors with a reliable count to help determine medication efficacy or other clinical endpoint. An ASIC that implements the EEG recording and seizure detection algorithm was designed and fabricated in a 0.18 μm CMOS process. The ASIC includes eight EEG channels and is designed to minimize the system's power and size. The result is a power-efficient analog front end that requires 2.75 μW per channel in diagnosis mode and 0.84 μW per channel in seizure counting mode. Both modes have an input referred noise of approximately 1.1 μVrms.


Frequency and significance of antibodies to chromatin in autoimmune hepatitis.
Our aims were to determine the frequency and prognostic implications of antibodies to chromatin in autoimmune hepatitis. Three hundred seventy-one serum samples from 172 patients were tested by enzyme-linked immunosorbent assay. Sixty-seven patients (39%) had antibodies to chromatin. Percent positivity was greater in men than women (58% vs 34%, P = 0.008), and seropositivity was associated with higher serum levels of gamma-globulin and immunoglobulin G. Antibodies to chromatin disappeared in 25 of 60 patients who were tested successively (42%), and they were more common in samples obtained during active than inactive disease (32% vs 19%, P = 0.01). Relapse after drug withdrawal occurred more often in seropositive patients (91% vs 66%, P = 0.002). We conclude that antibodies to chromatin occur commonly in autoimmune hepatitis, and they are associated with disease activity. Percent positivity is greater in men than women, and seropositivity identifies individuals who commonly relapse after drug withdrawal.


[Surgery of the extracranial cerebral vessels. (Indication and results)].
Surgery of the extracranial cerebral vessels nowadays under right indication and operative technique is one of the most satisfying areas of vascular surgery. Our present status of indication is shown in table 1; it should be pointed out that the classical indication is a lesion of the internal carotid artery with a Transitoric Ischemic Attack, but also in cases of progressive stroke with acute carotid occlusion the operation will be successful relentless of any time limit as long as the patient is conscious. For the extrathoracal bypass procedure we now prefer the subclavio-subclavian bypass to the formerly performed carotio-subclavian bypass.


Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants.
Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of approximately 10(3). Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution.


[Ureter and Wertheim's operation].
The surgery for carcinoma of the uterine cervix combined with radiotherapy, as carried out at present, includes a definite risk for the urinary tract. Not so much the immediate functional disorders, which always recede, except in rare cases of fibrous retracted bladder, but the lesions which occur in several varieties lead one to re-implant immediately the ureter. Fistulae and early stenosis (16 out of 1,500) should be treated in the same way. These late complications, such as obstruction (12 out of 1,500) raise de problem of their etiology. Usually they are due to recurrent carcinoma (9 out of 12), rarely due to pelvic fibrosis (3 out of 12). It is rare then for re-implantation to be possible. There remains either nephrectomy which is the simplest solution, or an urinary by-pass operation either using an ileal bladder or as a palliative measure, using a silastic prosthesis. Finally, the risk of advanced carconoma is without remedy, abstention from surgery may then be justified but one should, as far as possible, attempt to prevent these complications both from the surgical technical point of view and from the point of view of irradiation dosage.


Standard and advanced echocardiography in takotsubo (stress) cardiomyopathy: clinical and prognostic implications.
Echocardiography is frequently the initial noninvasive imaging modality used to assess patients with takotsubo cardiomyopathy (TTC). Standard transthoracic echocardiography can provide, even in the acute care setting, useful information about left ventricular (LV) morphology as well as regional and global systolic or diastolic function. It allows the differentiation of different LV morphologic patterns according to the localization of wall motion abnormalities. A "circumferential pattern" of LV myocardial dysfunction characterized by symmetric wall motion abnormalities involving the midventricular segments of the anterior, inferior, and lateral walls should be considered suggestive of TTC and included in the differential diagnosis of acute coronary syndromes. Moreover, advanced echocardiographic techniques, including speckle-tracking, myocardial contrast, and coronary flow studies, are providing mechanistic and pathophysiologic insights into this unique syndrome. Early identification of any potential complications (i.e., LV outflow tract obstruction, reversible moderate to severe mitral regurgitation, right ventricular involvement, thrombus formation, and cardiac rupture) are crucial for the management, risk stratification, and follow-up of patients with TTC. Because of the dynamic evolution of the syndrome, comprehensive serial echocardiographic examinations should be systematically performed. This review focuses on these aspects of imaging and the increasing understanding of the clinical and prognostic utility of echocardiography in TTC.


DNA driven self-assembly of micron-sized rods using DNA-grafted bacteriophage fd virions.
We have functionalized the sides of fd bacteriophage virions with oligonucleotides to induce DNA hybridization driven self-assembly of high aspect ratio filamentous particles. Potential impacts of this new structure range from an entirely new building block in DNA origami structures, inclusion of virions in DNA nanostructures and nanomachines, to a new means of adding thermotropic control to lyotropic liquid crystal systems. A protocol for producing the virions in bulk is reviewed. Thiolated oligonucleotides are attached to the viral capsid using a heterobifunctional chemical linker. A commonly used system is utilized, where a sticky, single-stranded DNA strand is connected to an inert double-stranded spacer to increase inter-particle connectivity. Solutions of fd virions carrying complementary strands are mixed, annealed, and their aggregation is studied using dynamic light scattering (DLS), fluorescence microscopy, and atomic force microscopy (AFM). Aggregation is clearly observed on cooling, with some degree of local order, and is reversible when temperature is cycled through the DNA hybridization transition.


Differential expression of melanoma associated antigens in acral lentiginous melanoma and in nodular melanoma lesions.
The reactivity in an avidin-biotin complex immunoperoxidase reaction with a large panel of anti-human melanoma associated antigen (MAA) and anti-HLA monoclonal antibodies of 24 primary and 11 metastatic acral lentiginous melanoma (ALM) lesions was compared to that of 12 primary and 12 metastatic nodular melanoma (NM) lesions. The expression of the membrane bound vitronectin receptor, Mr 110,000 MAA, Mr 97,000 MAA, and intercellular adhesion molecule-1 was significantly lower in both primary and metastatic ALM lesions than in their NM counterparts. Furthermore, primary ALM lesions displayed a significantly lower expression than primary NM lesions of the membrane bound high molecular weight melanoma associated antigen (HMW-MAA), Mr 110,000 MAA, Mr 100,000 MAA, 9-O-acetyl-GD3, GD2-GD3, and GD2, of the cytoplasmic monoclonal antibody 465.12 defined MAA and of transferrin receptor and of HLA-DQ and DP antigens; ALM metastases expressed a significantly lower level of carcinoembryonic antigen-MAA than NM metastases. These antigenic differences do not reflect an antigenic paucity of ALM cells, since ALM lesions express a higher level of T4-tyrosinase than NM lesions and a level of HLA Class I antigens similar to that of NM lesions. In view of the use of HMW-MAA, Mr 97,000 MAA, and GD3 in immunoscintigraphy and/or in immunotherapy, it is noteworthy that the three antigens are expressed in a similar high percentage of ALM metastases and of primary and metastatic NM lesions, while the HMW-MAA is expressed in a markedly lower percentage of primary ALM lesions than Mr 97,000 MAA and GD3. However, the degree of heterogeneity of HMW-MAA within a positive primary ALM lesion, as measured by the percentage of stained melanoma cells, is lower than that of Mr 97,000 MAA and GD3. The expression of the antigens investigated in ALM and NM lesions was not correlated with the presence of lymphocyte infiltrates, melanin content of melanoma cells, and epithelioid and spindle type of melanoma cells in the lesions. On the other hand, the survival of patients with ALM was inversely correlated with the expression of intercellular adhesion molecule 1 or HMW-MAA in their primary lesions. A potential role of HMW-MAA in the course of the disease is suggested by its significantly higher expression in metastatic than in primary ALM lesions.(ABSTRACT TRUNCATED AT 400 WORDS)


Structure of the oligonucleotide d(CGTATATACG) as a site-specific complex with nickel ions.
In this paper we explore the application of Ni2+to the crystallization of oligonucleotides. We have determined in this way the structure of a fully alternating (Y-R) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction. This is the first oligonucleotide crystal structure with an alternating 5'-(TA)3-3' central part. Alternating oligonucleotides have a particular interest since they often have a unique structure. In this case the general conformation is B-like with an alternating twist and an end-to-end interaction which involves terminal guanines. The crystal belongs to space group P41212 with a = b = 52.46, c = 101.49 A. This packing imposes a 90 degrees crossing of the symmetry related helices. This is a new way of packing for decamers. The oligonucleotide structure is characterized by the specific association with seven nickel ions, involving the N7 atom of every guanine. One of the Ni2+ions is shared between two guanines of symmetry related molecules. Until now no oligonucleotide has been crystallized in the presence of this metal ion. A novel C.A.T triplet structure has also been tentatively identified.


Improved time to disease progression in the brain in patients with melanoma brain metastases treated with concurrent delivery of radiosurgery and ipilimumab.
Background: To identify the optimal sequencing and timing of immunotherapy (IT) and stereotactic radiosurgery (SRS) for melanoma brain metastases (MBMs). Methods: The elapsed days between IT and SRS were correlated with local control (LC), regional brain control (RBC), time to CNS progression (TTPCNS), overall survival (OS), and radiation necrosis (RN). Logistic regression and Cox proportional models were used for statistical analysis. Results: Twenty-five patients with 58 MBMs underwent SRS and IT. Median follow-up was 22.7 mo (3.1-77.9 mo). A median of 2 SRS treatments of 21 Gy (range 16-24 Gy) and 4 cycles of Ipilimumab were delivered. SRS was delivered Before, After or Concurrently with IT in 9, 5, and 11 patients, respectively; 8/25 received SRS ≤30 d of IT and 17/25 were >30 d of IT. Median OS was 35.8 mo, 1- and 2-y OS was 83% and 64%, respectively, and LC was 94.8%. By timing, RBC and TTPCNS were significantly improved when SRS was delivered ≤30 d of IT (75% vs 23.5%, p = 0.03 and median not reached vs 5.7 mo, p = 0.02, respectively). By groups, Concurrent delivery improved TTPCNS (p = 0.04). The rate of RN was 20.7% (12/58 lesions) and RN was associated with improved OS (HR 0.21, p = 0.01). Conclusions: High OS was found for MBM treated with SRS and IT compared to historical reports. A significant association for improved RBC and TTPCNS was found when SRS was delivered concurrently and within 30 d of IT. Occurrence of RN was higher than SRS alone series but significantly associated with improved OS.


Conditional stomatal closure in a fern shares molecular features with flowering plant active stomatal responses.
Stomata evolved as plants transitioned from water to land, enabling carbon dioxide uptake and water loss to be controlled. In flowering plants, the most recently divergent land plant lineage, stomatal pores actively close in response to drought. In this response, the phytohormone abscisic acid (ABA) triggers signaling cascades that lead to ion and water loss in the guard cells of the stomatal complex, causing a reduction in turgor and pore closure. Whether this stimulus-response coupling pathway acts in other major land plant lineages is unclear, with some investigations reporting that stomatal closure involves ABA but others concluding that closure is passive. Here, we show that in the model fern Ceratopteris richardii active stomatal closure is conditional on sensitization by pre-exposure to either low humidity or exogenous ABA and is promoted by ABA. RNA-seq analysis and de novo transcriptome assembly reconstructed the protein-coding complement of the C. richardii genome, with coverage comparable to other plant models, enabling transcriptional signatures of stomatal sensitization and closure to be inferred. In both cases, changes in abundance of homologs of ABA, Ca2+, and ROS-related signaling components were observed, suggesting that the closure-response pathway is conserved in ferns and flowering plants. These signatures further suggested that sensitization is achieved by lowering the threshold required for a subsequent closure-inducing signal to trigger a response. We conclude that the canonical signaling network for active stomatal closure functioned in at least a rudimentary form in the stomata of the last common ancestor of ferns and flowering plants.


Epidemiology of resistance and phenotypic characterization of carbapenem resistance mechanisms in Klebsiella pneumoniae isolates at Sahloul University Hospital-Sousse, Tunisia.
To assess the prevalence of ESBL producing and carbapenem resistant Klebsiella pneumoniae isolated from in-come and out-come patients at Sahloul-university hospital. A retrospective study over a 3 years period (January 2012 and December 2014) focused on 2160 strains of Klebsiella pneumoniae. Statistical analysis was carried out using SPSS program. ESBL detection was performed using a double disc diffusion method and carbapenemase detection was realized by Rosco-Disk kit. A total of 2160 Klebsiella pneumoniae strains were isolated during the period of the study, 26.2% (n=566) were ESBL-producers and 15.8% (n=342) showed resistance to carbapenem. The wards most affected by these strains were basically urology and intensive care units. Eighty four percent of studied strains (203/241) were resistant to temocillin, which correlate with the production of a class D (OXA-48-like) carbapenemase and 7% (17/241) showed sensitivity to EDTA and dipicolinic acid, which indicate the production of metallo-enzyme. The rate of resistance to colistin remains low. Resistance of Enterobacteriaceae, including K. pneumoniae, to third generation cephalosporins (3rd GC) and carbapenem through the mechanism of ESBL and carbapenemases production is becoming increasingly worrying. This suggests a more rational use of antibiotics, as well as the rigorous application of hygiene measurement.


Necrotizing meningoencephalitis of Pug dogs associates with dog leukocyte antigen class II and resembles acute variant forms of multiple sclerosis.
Necrotizing meningoencephalitis (NME) is a disorder of Pug Dogs that appears to have an immune etiology and high heritability based on population studies. The present study was undertaken to identify a genetic basis for the disease. A genome-wide association scan with single tandem repeat (STR) markers showed a single strong association near the dog leukocyte antigen (DLA) complex on CFA12. Fine resolution mapping with 27 STR markers on CFA12 further narrowed association to the region containing DLA-DRB1, -DQA1 and, -DQB1 genes. Sequencing confirmed that affected dogs were more likely to be homozygous for specific alleles at each locus and that these alleles were linked, forming a single high risk haplotype. The strong DLA class II association of NME in Pug Dogs resembles that of human multiple sclerosis (MS). Like MS, NME appears to have an autoimmune basis, involves genetic and nongenetic factors, has a relatively low incidence, is more frequent in females than males, and is associated with a vascularly orientated nonsuppurative inflammation. However, NME of Pug Dogs is more aggressive in disease course than classical human MS, appears to be relatively earlier in onset, and involves necrosis rather than demyelination as the central pathobiologic feature. Thus, Pug Dog encephalitis (PDE) shares clinical features with the less common acute variant forms of MS. Accordingly, NME of Pug Dogs may represent a naturally occurring canine model of certain idiopathic inflammatory disorders of the human central nervous system.


LATERAL TARSAL STRIP TECHNIQUE IN CORRECTION OF EYELID ECTROPION AND ENTROPION.
Ectropion and entropion are eyelid malpositions associated with a number of unpleasant symptoms. One of several surgical methods that can correct these problems is referred to as the lateral tarsal strip technique. In this paper, we evaluate the postoperative results and quality of this technique. We also examine the complications associated with this surgical method. The study took place from April 2018 to April 2020 at the Military University Hospital in Prague. The operation was performed by two surgeons. The study included 43 eyes of 33 patients (17 women and 16 men), average age was 79 years. Before the operation, 23 eyes were affected by ectropion and 20 eyes affected by entropion, 35 of the 43 were cases of involutional origin. The study does not include patients who underwent other surgical techniques. There were no perioperative or postoperative complications in the entropion surgery group. Successful correction was achieved, and adverse symptoms disappeared in 91.3% of cases. Correct position of the eyelid was not achieved in two cases. For patients with ectropion, surgical correction was successful in 95% of cases. Ectropion persisted after surgery only in one patient. The lateral tarsal strip technique is a safe, reliable and highly effective surgical technique. Correction of eyelid malposition was achieved in more than 90% of cases. The vast majority of patients were satisfied and reported relief from preoperative symptoms.


Development of a new method for estimating visceral fat area with multi-frequency bioelectrical impedance.
Excessive visceral fat area (VFA) is a major risk factor in such conditions as cardiovascular disease. In assessing VFA, computed tomography (CT) is adopted as the gold standard; however, this method is cost intensive and involves radiation exposure. In contrast, the bioelectrical impedance (BI) method for estimating body composition is simple and noninvasive and thus its potential application in VFA assessment is being studied. To overcome the difference in obtained impedance due to measurement conditions, we developed a more precise estimation method by selecting the optimum body posture, electrode arrangement, and frequency. The subjects were 73 healthy volunteers, 37 men and 36 women, who underwent CT scans to assess VFA and who were measured for anthropometry parameters, subcutaneous fat layer thickness, abdominal tissue area, and impedance. Impedance was measured by the tetrapolar impedance method using multi-frequency BI. Multiple regression analysis was conducted to estimate VFA. The results revealed a strong correlation between VFA observed by CT and VFA estimated by impedance (r = 0.920). The regression equation accurately classified VFA > or = 100 cm(2) in 13 out of 14 men and 1 of 1 woman. Moreover, it classified VFA > or = 100 cm(2) or < 100 cm(2) in 3 out of 4 men and 1 of 1 woman misclassified by waist circumference (W) which was adopted as a simple index to evaluate VFA. Therefore, using this simple and convenient method for estimating VFA, we obtained an accurate assessment of VFA using the BI method.


Studies on an acquired inhibition of factor VIII induced by penicillin allergy.
An inhibitor of antihaemophilic globulin has been found in association with penicillin allergy. Inhibitor activity was detected after a severe reaction of penicillin. Neutralization studies showed the activity resided in an IgG globulin with kappa light chains. Experiments with insolubilized gammaglobulin demonstrated that the activity of the inhibitor was found in a specific penicillin antibody.


Can Early Omega-3 Fatty Acid Exposure Reduce Risk of Childhood Allergic Disease?
A causal link between increased intake of omega-6 (n-6) polyunsaturated fatty acids (PUFAs) and increased incidence of allergic disease has been suggested. This is supported by biologically plausible mechanisms, related to the roles of eicosanoid mediators produced from the n-6 PUFA arachidonic acid. Fish and fish oils are sources of long chain omega-3 (n-3) PUFAs. These fatty acids act to oppose the actions of n-6 PUFAs particularly with regard to eicosanoid synthesis. Thus, n-3 PUFAs may protect against allergic sensitisation and allergic manifestations. Epidemiological studies investigating the association between maternal fish intake during pregnancy and allergic outcomes in infants/children of those pregnancies suggest protective associations, but the findings are inconsistent. Fish oil provision to pregnant women is associated with immunologic changes in cord blood. Studies performed to date indicate that provision of fish oil during pregnancy may reduce sensitisation to common food allergens and reduce prevalence and severity of atopic eczema in the first year of life, with a possible persistence until adolescence. A recent study reported that fish oil consumption in pregnancy reduces persistent wheeze and asthma in the offspring at ages 3 to 5 years. Eating oily fish or fish oil supplementation in pregnancy may be a strategy to prevent infant and childhood allergic disease.


Aetiology of anorexia nervosa: from a "psychosomatic family model" to a neuropsychiatric disorder?
Eating disorders and, in particular, anorexia nervosa (AN) have morbidity and mortality rates that are among the highest of any mental disorders and are associated with significant functional impairment. More than 25 years ago, several researchers hypothesised that the prerequisite for the development of AN was a family process characterised by an overprotective and conflict-avoiding parent-child interaction. Family studies, however, suggest that AN is a complex genetic disorder that is likely expressed primarily by temperament and specific traits during childhood, including inhibition, perfectionism and harm avoidance. Recent studies have described an impaired flexibility and deficits in social cognition that are independent of body weight and the current state of the eating disorder, providing further evidence for a genetic component of AN. The physiological and psychological alterations and the increasing societal demands that occur during puberty may trigger onset. The starvation process itself is associated with severe alterations of central and peripheral metabolism, especially neuroendocrine and neurotransmitter changes, which are thought to affect the adolescent brain during the vulnerable period of neural restructuring. Long-standing malnutrition during adolescence and young adulthood associated with hormonal and neuropeptide dysfunctions may produce "biological scars" that maintain and accelerate the disorder and likely result in chronic mental disorders in adulthood as well as poor social functioning.


Risk of Parkinson's disease following zolpidem use: a retrospective, population-based cohort study.
To evaluate the influence of long-term zolpidem use on the incidence of developing Parkinson's disease. 2,961 subjects who used zolpidem for the first time longer than 3 months between 1998 and 2000 were identified in the National Health Insurance system of Taiwan. Subjects without a history of zolpidem use were randomly selected as a comparison cohort and frequency matched to zolpidem users based on age, sex, and index date. The diagnosis of Parkinson's disease was based on the criteria of the International Classification of Diseases, Ninth Revision, Clinical Modification. Its incidence until the end of 2009 was calculated and its hazard ratios (HRs) and 95% CIs were estimated using Cox proportional hazards regression models and Kaplan-Meier analysis. The overall incidence of Parkinson's disease was greater among zolpidem users than in the comparison cohort (HR = 1.88; 95% CI, 1.45-2.45). However, there was no difference in Parkinson's disease incidence between these 2 cohorts after 5 years of observation. The risk of Parkinson's disease increased with increasing zolpidem dose, with an HR of 0.70 for low-dose users (< 400 mg/y) and 2.94 for high-dose users (≥ 1,600 mg/y). The incidence of Parkinson's disease was greater in subjects using zolpidem only (HR = 2.35; 95% CI, 1.66-3.33) compared to those using benzodiazepines only (HR = 1.31; 95% CI, 0.91-1.90). By stratified analysis, zolpidem use with concurrent depression (HR = 4.79) increased the risk of Parkinson's disease compared to that of zolpidem users without concurrent depression. Zolpidem use might unmask preclinical Parkinson's disease, especially in patients with depression. However, large population-based, unbiased, randomized trials are warranted to confirm this finding.


Statistical approaches to detecting and analyzing tandem repeats in genomic sequences.
Tandem repeats (TRs) are frequently observed in genomes across all domains of life. Evidence suggests that some TRs are crucial for proteins with fundamental biological functions and can be associated with virulence, resistance, and infectious/neurodegenerative diseases. Genome-scale systematic studies of TRs have the potential to unveil core mechanisms governing TR evolution and TR roles in shaping genomes. However, TR-related studies are often non-trivial due to heterogeneous and sometimes fast evolving TR regions. In this review, we discuss these intricacies and their consequences. We present our recent contributions to computational and statistical approaches for TR significance testing, sequence profile-based TR annotation, TR-aware sequence alignment, phylogenetic analyses of TR unit number and order, and TR benchmarks. Importantly, all these methods explicitly rely on the evolutionary definition of a tandem repeat as a sequence of adjacent repeat units stemming from a common ancestor. The discussed work has a focus on protein TRs, yet is generally applicable to nucleic acid TRs, sharing similar features.


Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji).
The flavin-dependent enzyme pyranose 2-oxidase (P2Ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. P2Ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. Recently, it was shown that P2Ox is useful as bioelement in biofuel cells, replacing glucose oxidase (GOx), which traditionally is used in these applications. P2Ox offers several advantages over GOx for this application, e.g., its much broader substrate specificity. Because of this renewed interest in P2Ox, knowledge on novel pyranose oxidases isolated from organisms other than white-rot fungi, which represent the traditional source of this enzyme, is of importance, as these novel enzymes might differ in their biochemical and physical properties. We isolated and over-expressed the p2ox gene encoding P2Ox from the ectomycorrhizal fungus Lyophyllum shimeji. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in E. coli Rosetta 2. We obtained active, flavinylated recombinant P2Ox in yields of approximately 130 mg per L of medium. The enzyme was purified by a two-step procedure based on anion exchange chromatography and preparative native PAGE, yielding an apparently homogenous enzyme preparation with a specific activity of 1.92 U/mg (using glucose and air oxygen as the substrates). Recombinant P2Ox from L. shimeji was characterized in some detail with respect to its physical and catalytic properties, and compared to the well-characterised enzymes from Phanerochaete chrysosporium and Trametes multicolor. L. shimeji P2Ox shows properties that are comparable to those of P2Ox from white-rot fungal origin, and is in general characterised by lower K(m) and k(cat) values both for electron donor (sugar) as well as electron acceptor (ferrocenium ion, 1,4-benzoquinone, 2,6-dichloroindophenol). While L. shimeji P2Ox is the least thermostable of these three enzymes (melting temperature T(m) of 54.9 degrees C; half-life time of activity tau1/2 of 0.12 at 50 degrees C and pH 6.5), P. chrysosporium P2Ox showed remarkable thermostability with T(m) of 75.4 degrees C and tau1/2 of 96 h under identical conditions.


Low Incidence of Acute Antibody-Mediated Rejection after HLA Desensitization in Living Donor Kidney Transplant Recipients.
Desensitization allows the performance of human leukocyte antigen (HLA)-incompatible transplants. However, the incidence of acute rejection (AR) is high. This study aims to analyze the incidence of AR after transplantation with HLA-incompatible living donors in patients who underwent desensitization. Patients were immunosuppressed with tacrolimus, mycophenolic acid derivatives, and steroids after being desensitized with rituximab, plasma exchange, and/or immunoadsorption with specific cytomegalovirus immunoglobulins. A negative complement-dependent cytotoxicity or flow cytometry crossmatch and a donor-specific antibody titer < 1000 mean fluorescence intensity (MFI) were used to determine desensitization efficacy. A total of 36 patients underwent desensitization, and 27 (75%) were transplanted. After a follow-up of 58 ± 58 months (Min−Max: 0.13−169.5), five episodes of AR occurred: two antibody-mediated and three T-cell-mediated. No differences were found in baseline calculated panel-reactive antibodies (cPRA), class I or II MFI, number of antibodies, or Relative Intensity Scale (RIS) between AR and non-AR patients. Patients with antibody-mediated AR had higher cPRA (NS), MFI class I (p = 0.07) and class II (p = 0.006), and RIS (p = 0.01). The two patients with antibody-mediated AR and one patient with T-cell-mediated AR lost their grafts. In conclusion, the incidence of acute antibody-mediated rejection after desensitization was 7.4%, which occurred early post-transplantation in patients with high MFI and was associated with early graft loss.


Beyond confidence: Development of a measure assessing the 5C psychological antecedents of vaccination.
Monitoring the reasons why a considerable number of people do not receive recommended vaccinations allows identification of important trends over time, and designing and evaluating strategies to address vaccine hesitancy and increase vaccine uptake. Existing validated measures assessing vaccine hesitancy focus primarily on confidence in vaccines and the system that delivers them. However, empirical and theoretical work has stated that complacency (not perceiving diseases as high risk), constraints (structural and psychological barriers), calculation (engagement in extensive information searching), and aspects pertaining to collective responsibility (willingness to protect others) also play a role in explaining vaccination behavior. The objective was therefore to develop a validated measure of these 5C psychological antecedents of vaccination. Three cross-sectional studies were conducted. Study 1 uses factor analysis to develop an initial scale and assesses the sub-scales' convergent, discriminant, and concurrent validity (N = 1,445, two German convenience-samples). In Study 2, a sample representative regarding age and gender for the German population (N = 1,003) completed the measure for vaccination in general and for specific vaccinations to assess the potential need for a vaccine-specific wording of items. Study 3 compared the novel scale's performance with six existing measures of vaccine hesitancy (N = 350, US convenience-sample). As an outcome, a long (15-item) and short (5-item) 5C scale were developed as reliable and valid indicators of confidence, complacency, constraints, calculation, and collective responsibility. The 5C sub-scales correlated with relevant psychological concepts, such as attitude (confidence), perceived personal health status and invulnerability (complacency), self-control (constraints), preference for deliberation (calculation), and communal orientation (collective responsibility), among others. The new scale provided similar results when formulated in a general vs. vaccine-specific way (Study 2). In a comparison of seven measures the 5C scale was constantly among the scales that explained the highest amounts of variance in analyses predicting single vaccinations (between 20% and 40%; Study 3). The present studies are limited to the concurrent validity of the scales. The 5C scale provides a novel tool to monitor psychological antecedents of vaccination and facilitates diagnosis, intervention design and evaluation. Its short version is suitable for field settings and regular global monitoring of relevant antecedents of vaccination.


Two to Five-Year Outcomes of Total Ankle Arthroplasty with the Infinity Fixed-Bearing Implant: A Concise Follow-up of a Previous Report.
Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.


[Fever in intensive care: keep medications in mind at all times].
In two patients, men aged 35 and 69 years admitted postoperatively to the intensive care unit, fever of unknown origin developed. One had been admitted because aspiration was suspected. He had been treated immediately with amoxicillin and clavulanic acid. The other had undergone oesophageal excision and gastric reconstruction because of oesophageal carcinoma and had been subjected to antibiotic decontamination (amphotericin B, norfloxacine en fungizone). No cause for the fever was detected, but it quickly subsided after discontinuation of the amoxicillin-clavulanic acid and the norfloxacine, respectively. When encountering fever of unknown origin in intensive care patients it is always important to think of drug fever.


Surface roughness and wear behavior of occlusal splint materials made of contemporary and high-performance polymers.
With the development of a digital technology of computer-assisted manufacturing (CAD/CAM) and new age materials, the use of new types of occlusal splint is to consider. The aim of the present study was to evaluate the surface roughness (Ra) and wear behavior of different CAD/CAM materials against enamel antagonist through a simulated chewing test. A total of 75 specimens made from ethylene vinyl acetate (EVA), polymethyl methacrylate (PMMA), polycarbonate (PC), polyetheretherketone (PEEK), and polyethyleneterephthalate (PETG) as a control were polished to evaluate the Ra before loading by optical profilometry and further analyzed by scanning electron microscopy (SEM). Specimens of each group were subjected to thermomechanical fatigue loading in a chewing simulator (60000 cycles at 49 N with 5-55 °C thermocycling). The wear volume loss and change in Ra of each specimen after the simulated chewing were analyzed. One-way ANOVA, paired samples t test, and Pearson correlation analysis were performed for statistical analyzes. The result showed that the volume loss and Ra varied among the materials tested. EVA exhibited the greatest amount of Ra and volume loss (p < 0.001), while PEEK had the lowest values for both (p < 0.001). In terms of volume loss, there was no significant difference between PC and PMMA (p > 0.05). SEM investigations revealed different wear behaviors, especially in EVA. As PEEK showed significantly more favorable results, PEEK splints should be considered as a new therapeutic option for occlusal splint.


Requirements of the membrane proximal tyrosine and dileucine-based sorting signals for efficient transport of the subtype C Vpu protein to the plasma membrane and in virus release.
Previously, we showed that the Vpu protein from HIV-1 subtype C is more efficiently transported to the cell surface than the well studied subtype B Vpu (Pacyniak et al., 2005) and that a SHIV expressing the subtype C Vpu exhibited a decreased rate of CD4+ T cell loss following inoculation in macaques (Hill et al., 2008). In this study, we examined the role of overlapping tyrosine-based (YXXPhi) and dileucine-based ([D/E]XXXL[L/I]) motifs in the membrane proximal region of the subtype C Vpu (EYRKLL) in Vpu intracellular transport, CD4 surface expression and virus release from the cell surface. We constructed three site-directed mutants of the subtype C vpu and fused these genes to the gene for enhanced green fluorescent protein (EGFP). The first mutation made altered the tyrosine (EARKLL; VpuSCEGFPY35A), the second altered the dileucine motif (EYRKLG; VpuSCEGFPL39G), and the third contained both amino acid substitutions (EARKLG; VpuSCEGFPYL35,39AG) in this region of the Vpu protein. The VpuSCEGFPY35A protein was transported to the cell surface similar to the unmodified VpuSCEGFP1 while VpuSCEGFPL39G was expressed at the cell surface at significantly reduced levels. The VpuSCEGFPYL35,39AG was found to have an intermediate level of cell surface expression. All three mutant Vpu proteins were analyzed for the ability to prevent cell surface expression of CD4. We found that both single mutants did not significantly effect CD4 surface expression while the double mutant (VpuSCEGFPYL35,39AG) was significantly less efficient at preventing cell surface CD4 expression. Chimeric simian human immunodeficiency viruses were constructed with these mutations in vpu (SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG). Our results indicate that SHIVSCVpuL39G replicated much more efficiently and was much more cytopathic than SHIVSCVpu. In contrast, SHIVSCVpuY35A and SHIVSCVpuYL35,39AG replicated less efficiently when compared to the parental SHIVSCVpu. Taken together, these results show for the first time that the membrane proximal tyrosine-based sorting motif in the cytoplasmic domain of Vpu is essential for efficient virus release. These results also indicate that the dileucine-based sorting motif affects the intracellular trafficking of subtype C Vpu proteins, virus replication, and release.


Induction of a local anti-IpaC antibody response in mice by use of a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier.
The capacity of Shigella flexneri to act as a delivery system for stimulation of local immunity was investigated by use of an S. Flexneri 2a vaccine candidate (SC602). This vaccine strain was constructed by deletion of virulence genes responsible for dissemination (icsA) and survival (iuc:iut) of bacteria within the colonic mucosa. Among the most immunogenic S. flexneri 2a proteins inducing a local immunoglobulin A antibody response, the IpaC invasion was selected as a potential protein carrier. Overexpression of IpaC from a plasmid in trans within SC602 (SC602/pIpaC) was shown to be required for the induction of optimal anti-IpaC antibody response in the murine pulmonary infection model. A weak anti-IpaC antibody response was obtained after intranasal inoculations with SC602/pIpaC live bacteria. This response was enhanced by combining systemic priming with SC602/pIpaC killed bacteria and local boosting with SC602/pIpaC live bacteria. These results suggest that naive B cells primed systemically could account for local antibody production. This immunization protocol will allow further evaluation of the immunogenicity of IpaC hybrid proteins expresses in SC602.


Preparation of Cell Wall Antigens of Staphylococcus aureus.
Cell walls were prepared from Staphylococcus aureus strains Copenhagen and 263 by high-speed mixing in the presence of glass beads followed by differential centrifugation. Insoluble peptidoglycan complexes were derived from cell walls by extraction of teichoic acid with 10% trichloroacetic acid. Intact teichoic acid was prepared from each strain by digestion of cell walls with lysostaphin and isolated by column chromatography. Soluble glycopeptide (peptidoglycan in which only the glycan has been fragmented) and the stable complex of teichoic acid with glycopeptide were prepared by digestion of cell walls with Chalaropsis B endo-N-acetylmuramidase and were separated by column chromatography. Amino acid and amino sugar contents of walls and subunits of walls were comparable to those reported by others.


Results of potency tests of a vaccine against Cl. botulinum type C by different methods.
The potency of vaccine against Cl. botulinum type C was tested comparatively in mice, guinea pigs and mink, using the test methods employed in the United States, Great Britain and Sweden. Additionally the capacity to withstand challenge to toxin in ferrets was investigated. All species of laboratory animals proved to be protected against high toxin doses. On the other hand the humoral antitoxin titer of the immunized animals was low after one injection. After a second injection or after challenge with toxin the titer increased considerably.


Identification of subtypes in subjects with mild-to-moderate airflow limitation and its clinical and socioeconomic implications.
The purpose of this study was to identify subtypes in patients with mild-to-moderate airflow limitation and to appreciate their clinical and socioeconomic implications. Subjects who were aged ≥20 years and had forced expiratory volume in 1 second (FEV1) ≥60% predicted and FEV1/forced vital capacity <0.7 were selected from the fourth Korea National Health and Nutrition Examination Survey (KNHANES) in 2007-2012. The data were merged to the National Health Insurance reimbursement database during the same period. k-Means clustering was performed to explore subtypes. For clustering analysis, six key input variables - age, body mass index (BMI), FEV1% predicted, the presence or absence of self-reported wheezing, smoking status, and pack-years of smoking - were selected. Among a total of 2,140 subjects, five groups were identified through k-means clustering, namely putative "near-normal (n=232)," "asthmatic (n=392)," "chronic obstructive pulmonary disease (COPD) (n=37)," "asthmatic-overlap (n=893)," and "COPD-overlap (n=586)" subtypes. Near-normal group showed the oldest mean age (72±7 years) and highest FEV1 (102%±8% predicted), and asthmatic group was the youngest (46±9 years). COPD and COPD-overlap groups were male predominant and all current or ex-smokers. While asthmatic group had the lowest prescription rate despite the highest proportion of self-reported wheezing, COPD, asthmatic-overlap, and COPD-overlap groups showed high prescription rate of respiratory medicine. Although COPD group formed only 1.7% of total subjects, they showed the highest mean medical cost and health care utilization, comprising 5.3% of the total medical cost. When calculating a ratio of total medical expense to household income, the mean ratio was highest in the COPD group. Clinical and epidemiological heterogeneities of subjects with mild-to-moderate airflow limitation and a different level of health care utilization by each subtype are shown. Identification of a subtype with high health care demand could be a priority for effective utilization of limited resources.


Incidence of and Risk Factors and Reinterventions for Post-Pancreatoduodenectomy Hemorrhage: Retrospective Analysis.
To analyze the incidence of and risk factors for post-pancreatoduodenectomy (PD) hemorrhage (PPH) and to evaluate the outcomes of reinterventions for PPH. All PDs between January 2009 and December 2014 were retrospectively evaluated. PPH was evaluated according to the criteria of the International Study Group of Pancreatic Surgery. Both univariate and multivariate analyses of risk factors for PPH and mortality were performed. Reinterventions were also evaluated. Of the 1,056 PDs during the study period, 78 (7.4%) developed PPH, including 36 with grade B and 42 with grade C. Of these 78 patients, 24 (30.8%) died of PPH-related causes. Multivariate analysis showed that older age, higher total bilirubin concentration, and postoperative pancreatic fistula (POPF) were independent risk factors for PPH. Patients who died of PPH were significantly older and had lower preoperative hemoglobin and albumin concentrations than patients who did not die of PPH. Of the 78 patients with PPH, 58 underwent reintervention, including 27 who underwent angiography, 24 who underwent endoscopy, 24 who underwent re-laparotomy, and 15 who underwent more than one reintervention. Older age, total bilirubin, and POPF are independent risk factors for PPH. Higher mortality are associated with advanced PPH and poor nutritional conditions.