Dynamic computed tomography is useful for prediction of pathological grade in pancreatic neuroendocrine neoplasm.
Pathological grading is important in defining the therapeutic strategy in pancreatic neuroendocrine neoplasm (PNEN) but is difficult for unresectable cases. Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) is useful in the diagnosis of PNEN, but its usefulness for pathological grading is not well established. No studies have examined the diagnostic ability of dynamic computed tomography (CT) for pathological grading of PNEN. We investigated the usefulness of EUS-FNA and dynamic CT in the diagnosis and pathological grading of PNEN. In this retrospective study, 39 PNEN patients finally diagnosed via EUS-FNA and/or surgical resection underwent dynamic CT. Pathological samples were diagnosed based on WHO2010; staging was based on the European Neuroendocrine Tumor Society classification. The proportion of the quantification value in the tumor to the pancreatic parenchyma in arterial phase was defined as the CT ratio. Immunohistochemical staining with CD31 was performed to evaluate microvessel density (MVD). We evaluated the relationship between pathological grade, CT ratio, and MVD. By using EUS-FNA, 35 of 39 (90%) cases were diagnosed as PNEN. As for pathological grade, 15 of 35 (43%) cases could be identified correctly. CT ratio could predict pathological Grade 3 disease. The sensitivity, specificity, and diagnostic accuracy were 100%, 94%, and 95%. MVD was significantly correlated with CT ratio (r = 0.83, P < 0.0001) and pathological grade (P = 0.0074). Computed tomography ratio has a relationship with pathological grade in PNEN, which would help decide therapeutic strategy in unresectable cases and cases in which pathological grading is difficult.


The phenotype of gastric mucosa coexisting with Barrett's oesophagus.
Barrett's oesophagus complicates the gastro-oesophageal acid reflux. Helicobacter pylori infection, particularly with cagA positive strains, induces inflammatory/atrophic lesions of the gastric mucosa, which may impair acid output. No systematic study has investigated the phenotype of the gastric mucosa coexisting with Barrett's oesophagus. This study was designed to identify the phenotype of gastric mucosa associated with Barrett's oesophagus. In this retrospective case control study, the phenotype of the gastric mucosa was histologically characterised in 53 consecutive patients with Barrett's oesophagus and in 53 (sex and age matched) non-ulcer dyspeptic controls. Both patients and controls underwent extensive sampling of the gastric mucosa (two antral, one incisural, and two oxyntic biopsies). Intestinal metaplasia (IM) was categorised (type I, complete IM; types II and III, incomplete IM) by the high iron diamine stain; cagA status was ascertained by genotyping. Helicobacter pylori was present in 19 of the 53 patients with Barrett's oesophagus and in 30 of the 53 controls (p < 0.02); eight of the 19 patients with Barrett's oesophagus and 28 of the 35 controls harboured cagA positive H pylori (p < 0.03). The histological severity of non-atrophic gastritis detected in the controls was significantly higher than that detected in the patients with Barrett's oesophagus (p < 0.0001). Multifocal atrophic gastritis was present in 4% of the patients with Barrett's oesophagus and in 23% of controls (p < 0.01). The odds ratio for the association between multifocal atrophic gastritis and Barrett's oesophagus was 0.20 (95% confidence interval, 0.006 to 0.60). Gastric IM was detected in 13.2% of the patients with Barrett's oesophagus and in 30.1% of the controls (p < 0.03). Type III IM at the gastric mucosa was only detected among controls. Barrett's oesophagus is associated with a low prevalence of H pylori cagA positive infection and multifocal atrophic gastritis. This pathobiological pattern is considered to be associated with a low risk of distal gastric cancer.


Species-specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces.
Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.


Potent activation of mouse macrophages by recombinant interferon-gamma.
The ability of recombinant interferon-gamma (IFN-gamma) to activate mouse macrophages was investigated. The use of recombinant IFN-gamma has the advantage of being devoid of contaminating lymphokines. Two preparations of IFN-gamma were utilized, one which was not glycosylated and which was highly purified from Escherichia coli another which was glycosylated and which was from transfected COS-7 monkey cells. Both preparations of recombinant IFN-gamma activated murine macrophages to kill lymphoma and melanoma tumor targets, suggesting that glycosylation of the protein or the presence of other mammalian proteins is not essential for activation. Significant levels of cytolytic activity were induced from IFN-gamma (1 to 10 units/ml). This activity was undiminished by treatment of the IFN-gamma preparations with polymixin B at doses which neutralized endotoxin (50 micrograms/ml). Similarly, IFN-gamma, at low concentrations, induced an inhibition of migration by macrophages. Based on antiviral activity, IFN-gamma was shown to be 100 to 1000 times more potent than was IFN-beta as a macrophage-activating agent. Taken together, these results demonstrate that murine IFN-gamma is a macrophage-activating factor which is effective at physiological concentrations. Of particular interest is the observation that the nonglycosylated E. coli-derived IFN-gamma is active and therefore may be of value for therapeutic studies, since it can be easily produced in large amounts.


Thermal tolerance and acclimation capacity in the European common frog (Rana temporaria) change throughout ontogeny.
Phenotypic plasticity may allow ectotherms with complex life histories such as amphibians to cope with climate-driven changes in their environment. Plasticity in thermal tolerance (i.e., shifts of thermal limits via acclimation to higher temperatures) has been proposed as a mechanism to cope with warming and extreme thermal events. However, thermal tolerance and, hence, acclimation capacity, is known to vary with life stage. Using the common frog (Rana temporaria) as a model species, we measured the capacity to adjust lower (CTmin ) and upper (CTmax ) critical thermal limits at different acclimation temperatures. We calculated the acclimation response ratio as a metric to assess the stage-specific acclimation capacity at each of seven consecutive ontogenetic stages and tested whether acclimation capacity was influenced by body mass and/or age. We further examined how acclimation temperature, body mass, age, and ontogenetic stage influenced CTmin and CTmax . In the temperate population of R. temporaria that we studied, thermal tolerance and acclimation capacity were affected by the ontogenetic stage. However, acclimation capacity at both thermal limits was well below 100% at all life stages tested. The lowest and highest acclimation capacity in thermal limits was observed in young and late larvae, respectively. The relatively low acclimation capacity of young larvae highlights a clear risk of amphibian populations to ongoing climate change. Ignoring stage-specific differences in thermal physiology may drastically underestimate the climate vulnerability of species, which will hamper successful conservation actions.


Ryanodine and the calcium content of cardiac SR assessed by caffeine and rapid cooling contractures.
The ability of rabbit ventricular muscle sarcoplasmic reticulum (SR) to accumulate and retain Ca during Na-free perfusion was assessed using caffeine contractures and rapid cooling contractures (RCC). Muscles were exposed to a Na-free medium for 15 min, during which time a transient contracture developed and relaxed back to the resting tension level. Muscles were then either exposed to 20 mM caffeine or rapidly cooled to less than 5 degrees C, both of which produced large contractures. The magnitudes of those contractures are believed to be a relative index of SR Ca content. Reduction of extracellular [Ca] from 2 to 0.2 mM did not significantly alter the magnitude of either Na-free caffeine contractures or RCC. These are not the maximum contractures that can be obtained. The possibility that low extracellular [Ca] ([Ca]o) may increase passive Ca permeability is suggested in explanation of this effect. After equilibration with 100 nM ryanodine, both Na-free caffeine contractures and RCC are virtually eliminated. This suggests that even if the SR could accumulate Ca during the initial Na-free exposure in the presence of ryanodine, it could not retain that Ca in Na-free medium. It is proposed that the sarcolemmal Ca pump can extrude Ca from the cells at a rate sufficient to deplete the ryanodine-treated SR. When removal of Na was accompanied by increase of [K]o (to 12, 20, or 30 mM), the Na-free RCC were enhanced. Increasing [K]o during Na-free superfusion in the presence of ryanodine resulted in demonstrable RCC that were graded with [K] and completely suppressed by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrasound comparison of external and internal neck anatomy with the LMA Unique.
Internal neck anatomy landmarks and their relation after placement of an extraglottic airway devices have not been studied extensively by the use of ultrasound. Based on our group experience with external landmarks as well as internal landmarks evaluation with other techniques, we aimed use ultrasound to analyze the internal neck anatomy landmarks and the related changes due to the placement of the Laryngeal Mask Airway Unique. Observational pilot investigation. Non-obese adult patients with no evidence of airway anomalies, were recruited. External neck landmarks were measured based on a validated and standardized method by tape. Eight internal anatomical landmarks, reciprocal by the investigational hypothesis to the external landmarks, were also measured by ultrasound guidance. The internal landmarks were re-measured after optimal placement and inflation of the extraglottic airway devices cuff Laryngeal Mask Airway Unique. Six subjects were recruited. Ultrasound measurements of hyoid-mental distance, thyroid-cricoid distance, thyroid height, and thyroid width were found to be significantly (p < 0.05) overestimated using a tape measure. Sagittal neck landmark distances such as thyroid height, sternal-mental distance, and thyroid-cricoid distance significantly decreased after placement of the Laryngeal Mask Airway Unique. The laryngeal mask airway Unique resulted in significant changes in internal neck anatomy. The induced changes and respective specific internal neck anatomy landmarks could help to design devices that would modify their shape accordingly to areas of greatest displacement. Also, while external neck landmark measurements overestimate their respective internal neck landmarks, as we previously reported, the concordance of each measurement and their respective conversion factor could continue to be of help in sizing extraglottic airway devices. Due to the pilot nature of the study, more investigations are warranted.


A potential role for dendritic cell/macrophage-expressing DPP4 in obesity-induced visceral inflammation.
Dipeptidyl peptidase-4 (DDP4) inhibitors target the enzymatic degradation of incretin peptides and represent a major advance in the treatment of type 2 diabetes. DPP4 has a number of nonenzymatic functions that involve its interaction with adenosine deaminase (ADA) and other extracellular matrix proteins. Here, we assessed the nonenzymatic role of DPP4 in regulating dendritic cell (DC)/macrophage-mediated adipose inflammation in obesity. Both obese humans and rodents demonstrated increased levels of DPP4 expression in DC/macrophage cell populations from visceral adipose tissue (VAT). The DPP4 expression increased during monocyte differentiation to DC/macrophages and with lipopolysaccharide (LPS)-induced activation of DC/macrophages. The DPP4 colocalized with membrane-bound ADA on human DCs and enhanced the ability of the latter to stimulate T-cell proliferation. The DPP4 interaction with ADA in human DC/macrophages was competitively inhibited by the addition of exogenous soluble DPP4. Knockdown of DPP4 in human DCs, but not pharmacologic inhibition of their enzymatic function, significantly attenuated the ability to activate T cells without influencing its capacity to secrete proinflammatory cytokines. The nonenzymatic function of DPP4 on DC may play a role in potentiation of inflammation in obesity by interacting with ADA. These findings suggest a novel role for the paracrine regulation of inflammation in adipose tissue by DPP4.


Functional anatomy of neural circuits regulating fear and extinction.
The memory of fear extinction is context dependent: fear that is suppressed in one context readily renews in another. Understanding of the underlying neuronal circuits is, therefore, of considerable clinical relevance for anxiety disorders. Prefrontal cortical and hippocampal inputs to the amygdala have recently been shown to regulate the retrieval of fear memories, but the cellular organization of these projections remains unclear. By using anterograde tracing in a transgenic rat in which neurons express a dendritically-targeted PSD-95:Venus fusion protein under the control of a c-fos promoter, we found that, during the retrieval of extinction memory, the dominant input to active neurons in the lateral amygdala was from the infralimbic cortex, whereas the retrieval of fear memory was associated with greater hippocampal and prelimbic inputs. This pattern of retrieval-related afferent input was absent in the central nucleus of the amygdala. Our data show functional anatomy of neural circuits regulating fear and extinction, providing a framework for therapeutic manipulations of these circuits.


Stroke volume during exercise in cystic fibrosis.
Previous studies comparing cardiac output (Q) and stroke volume (SV) between cystic fibrosis (CF) patients and control subjects have shown conflicting results: some found lower SV in CF patients with severe airflow limitation, and others showed no difference between CF and control subjects. Methodologic problems could explain these discrepant findings. The aim of this study was to better characterize Q and SV with exercise in CF patients with mild as well as severe airflow obstruction. Subjects included 18 CF patients with FEV1 ranging from 28 to 80% of predicted without pulmonary hypertension, and 16 matched control subjects. Cardiac output was measured at three levels of upright cycle exercise using the indirect Fick (CO2) method with blood gas sampling. Q on exercise was similar among control and CF subjects. SV was lower in CF patients, particularly those with FEV1 < or = 55% predicted, than in control subjects. Stepwise regression of SV on height, percent ideal body weight, and FEV1 showed a significant effect of relative underweight on SV. Despite this, well-nourished patients with FEV1 56 to 80% of predicted also had lower SV. As these findings were consistent across the range of severity of lung disease and age, even in the absence of malnutrition, they imply that another mechanism accounts for SV limitation during exercise in CF.


Uncoupling skeletal and connective tissue patterning: conditional deletion in cartilage progenitors reveals cell-autonomous requirements for Lmx1b in dorsal-ventral limb patterning.
Integration of muscle, connective tissue and skeletal patterning during development is essential for proper functioning of the musculoskeletal system. How this integration is achieved is poorly understood. There is ample evidence suggesting that skeletal pattern is programmed autonomously, whereas muscle pattern is, for the most part, programmed non-cell-autonomously. Connective tissues depend upon both muscle and skeletal tissues for their proper survival and development. Here, we employed a novel approach to dissect the coordination of musculoskeletal patterning during mouse limb development. Using both conditional gain- and loss-of-function approaches, we selectively deleted or activated the LIM-homeodomain transcription factor Lmx1b in skeletal progenitors using a Sox9-Cre knock-in allele. As Lmx1b is both necessary and sufficient to specify dorsal pattern, this approach allowed us to investigate the effect of selectively deleting or activating Lmx1b in skeletal progenitors on muscle, connective and skeletal tissues during limb development. Our results indicate that whereas Lmx1b activity is required autonomously in skeletal progenitors to direct dorsal pattern, loss or gain of Lmx1b activity in skeletal progenitors has no effect on muscle or connective tissue patterning. Hence, we show for the first time that skeletal and connective tissue patterning can be uncoupled, indicating a degree of autonomy in the formation of the musculoskeletal system.


Idiopathic scoliosis and spondylolysis in the female athlete. Tips for treatment.
Idiopathic scoliosis and spondylolysis can be common back problems in female athletes. Diagnosis and treatment can be difficult. With the notable trend toward increasing participation of women and girls in organized sports, it is necessary to know which sports carry additional risks for participants to have these two conditions develop and to determine treatment modalities. In general, idiopathic scoliosis is more prevalent in females and even may be higher in the athletes. Treatment options may include observation, the use of a brace, and surgery. In determining treatment, the type of sport and caliber of athlete must be considered in conjunction with the severity of the curve. Spondylolysis or a stress fracture of the posterior vertebral elements can be a common cause of back pain in an athlete. In many sports that are dominated by females (gymnastics, dancing, figure skating), the athletes carry a high risk of having spondylolysis or a stress fracture. Knowing the risk factors permits precise diagnosis and appropriate treatment. Treatment options include the use of a brace and surgery. In the current study, an extensive review of the literature in conjunction with the extensive experience of a well-established sports medicine clinic at the authors' institution is presented.


The Safety and Effectiveness of a Novel Annular Keratopigmentation Method: A Case Report.
We investigated the safety and effectiveness of a novel aesthetic femtosecond-assisted annular keratopigmentation technique. A 21-year-old female patient in good general and ophthalmological health with the wish to change the colour of her eyes was treated with a femtosecond-assisted annular keratopigmentation technique. Pigment was inserted in a channel in the cornea (external diameter 9.3 mm; internal diameter 5.5 mm) created with a femtosecond laser at a depth of 225 μm. Eight months post-operatively, there were no signs of leakage, diffusion, inflammation or any other detrimental effects on the cornea both objectively and subjectively. This procedure is a promising safe and effective option for those who want annular keratopigmentation for aesthetic reasons.


The Influence of Starch Origin on the Properties of Starch Films: Packaging Performance.
Starch films can be used as materials for food packaging purposes. The goal of this study is to compare how the starch origin influence the selected starch film properties. The films were made from various starches such as that from maize, potato, oat, rice, and tapioca using 50%w of glycerine as a plasticizer. The obtained starch-based films were made using the well-known casting method from a starch solution in water. The properties of the films that were evaluated were tensile strength, water vapour transition rate, moisture content, wettability, and their surface free energy. Surface free energy (SFE) and its polar and dispersive components were calculated using the Owens-Wendt-Rabel-Kaelbe approach. The values of SFE in the range of 51.64 to 70.81 mJ∙m-2 for the oat starch-based film and the maize starch-based film. The films revealed worse mechanical properties than those of conventional plastics for packaging purposes. The results indicated that the poorest tensile strength was exhibited by the starch-based films made from oat (0.36 MPa) and tapioca (0.78 MPa) and the greatest tensile strength (1.49 MPa) from potato.


Patient and physician preferences for the treatment of chronic hepatitis C virus infections: does the perspective matter?
The objectives of this study were to identify and quantify the factors driving patient and physician preferences for treatments of genotype 1 hepatitis C virus infection in the UK. A web survey was conducted, including 100 patients (50 treatment-naive and 50 treatment-experienced patients) and 50 physicians (gastroenterologists/ hepatologists and infectious disease specialists). A discrete-choice experiment was conducted to elicit the participants' preferences on the basis of seven attributes with four levels each: efficacy, that is probability of reaching sustained virologic response, treatment duration, treatment convenience (i.e. number of pills and/or injections), gastrointestinal problems, anaemia, dermatological problems and neuropsychological problems. The statistical analysis applied a mixed logit model to estimate preference weights and relative importance scores. Results indicated that the sustained virologic response rate was the most important attribute to participants. Physicians placed an even greater weight on the efficacy of treatments with a relative importance score of 9.33 [95% confidence interval: (6.93-11.91)], as compared with 6.16 [95% confidence interval: (4.34-8.15)] for patients. Neuropsychological problems ranked second for patients and physicians, and were more important to treatment-naive patients than to treatment-experienced patients or physicians. Gastrointestinal problems, anaemia and dermatological problems were of minor importance to all participants. These findings may be explained by the improvement in the management of physical adverse reactions over the last few years, thus making treatment easier to tolerate. This study is the first conjoint analysis assessing and comparing the preferences of patients and physicians in hepatitis C virus.


Active immunotherapy for chronic diseases.
With the effective control of infectious diseases in many parts of the world, chronic, non-communicable diseases have become the major cause of death and disability. Monoclonal antibodies (mAbs) have become an important class of drugs for the treatment of such diseases. Nevertheless, mAbs suffer from major shortcomings in a chronic setting: most notably, generation of anti-antibodies and high cost of goods. Here, we discuss a novel approach to treat chronic diseases based on active rather than passive immunization and contrast the 2 treatment modalities to highlight their respective advantages and disadvantages.


Toward an evolutionary definition of cheating.
The term "cheating" is used in the evolutionary and ecological literature to describe a wide range of exploitative or deceitful traits. Although many find this a useful short hand, others have suggested that it implies cognitive intent in a misleading way, and is used inconsistently. We provide a formal justification of the use of the term "cheat" from the perspective of an individual as a maximizing agent. We provide a definition for cheating that can be applied widely, and show that cheats can be broadly classified on the basis of four distinctions: (i) whether cooperation is an option; (ii) whether deception is involved; (iii) whether members of the same or different species are cheated; and (iv) whether the cheat is facultative or obligate. Our formal definition and classification provide a framework that allow us to resolve and clarify a number of issues, regarding the detection and evolutionary consequences of cheating, as well as illuminating common principles and similarities in the underlying selection pressures.


Development of a multidisciplinary, multicampus subspecialty practice in endocrine cancers.
Relative to more abundant neoplasms, endocrine cancers have been historically neglected, yet their incidence is increasing. We therefore sought to build interest in endocrine cancers, improve physician experience, and develop innovative approaches to treating patients with these neoplasms. Between 2005 and 2010, we developed a multidisciplinary Endocrine Malignancies Disease Oriented Group involving all 3 Mayo Clinic campuses (Rochester, Minnesota; Jacksonville, Florida; and Scottsdale, Arizona). In response to higher demand at the Rochester campus, we sought to develop a Subspecialty Tumor Group and an Endocrine Malignancies Tumor Clinic within the Division of Medical Oncology. The intended groups were successfully formed. We experienced difficulty in integration of the Mayo Scottsdale campus resulting from local uncertainty as to whether patient volumes would be sufficient to sustain the effort at that campus and difficulty in developing enthusiasm among clinicians otherwise engaged in a busy clinical practice. But these obstacles were ultimately overcome. In addition, with respect to the newly formed medical oncology subspecialty endocrine malignancies group, appointment volumes quadrupled within the first year and increased 7 times within 2 years. The number of active therapeutic endocrine malignancies clinical trials also increased from 1 in 2005 to 5 in 2009, with all 3 Mayo campuses participating. The development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.


Efficacy and Safety of Peginterferon Alfa-2a (40KD) in Children With Chronic Hepatitis B: The PEG-B-ACTIVE Study.
Children with chronic hepatitis B (CHB) represent an area of unmet medical need, attributed to increased lifetime risk of CHB sequelae and limited therapeutic options compared with adult CHB patients. The PEG-B-ACTIVE (NCT01519960) phase III study evaluated peginterferon (PegIFN) alfa-2a treatment in children aged 3 to <18 years with CHB. A total of 161 hepatitis B e antigen (HBeAg)-positive immune-active patients without advanced fibrosis (AF)/cirrhosis were randomized (2:1) to PegIFN alfa-2a (Group A, n = 101) or no treatment (Group B, n = 50); patients with AF were assigned to PegIFN alfa-2a (Group C, n = 10). PegIFN alfa-2a was administered for 48 weeks by body surface area (BSA) category, based on 180 μg/1.73 m2 . HBeAg seroconversion rates at 24 weeks posttreatment were significantly higher in Group A (25.7% vs. 6%; P = 0.0043), as were the rates of hepatitis B surface antigen (HBsAg) clearance (8.9% vs. 0%; P = 0.03), hepatitis B virus (HBV) DNA <2,000 IU/mL (28.7% vs. 2.0%; P < 0.001) or undetectable (16.8% vs. 2.0%; P = 0.0069), and alanine aminotransferase (ALT) normalization (51.5% vs. 12%; P < 0.001). Safety, including incidence of ALT flares and neutropenia, was comparable to the established PegIFN alfa-2a profile in HBV-infected adults or hepatitis C virus-infected children. Changes in growth parameters were minimal during treatment and comparable to those in untreated patients. Safety and efficacy outcomes in Group C were in line with Group A. Conclusion: PegIFN alfa-2a treatment of children in the immune-active phase of CHB was efficacious and well tolerated, and associated with higher incidence of HBsAg clearance than in adults. This represents an important advance to the treatment options for children with CHB.


Shrub canopies influence soil temperatures but not nutrient dynamics: An experimental test of tundra snow-shrub interactions.
Shrubs are the largest plant life form in tundra ecosystems; therefore, any changes in the abundance of shrubs will feedback to influence biodiversity, ecosystem function, and climate. The snow-shrub hypothesis asserts that shrub canopies trap snow and insulate soils in winter, increasing the rates of nutrient cycling to create a positive feedback to shrub expansion. However, previous work has not been able to separate the abiotic from the biotic influences of shrub canopies. We conducted a 3-year factorial experiment to determine the influences of canopies on soil temperatures and nutrient cycling parameters by removing ∼0.5 m high willow (Salix spp.) and birch (Betula glandulosa) shrubs, creating artificial shrub canopies and comparing these manipulations to nearby open tundra and shrub patches. Soil temperatures were 4-5°C warmer in January, and 2°C cooler in July under shrub cover. Natural shrub plots had 14-33 cm more snow in January than adjacent open tundra plots. Snow cover and soil temperatures were similar in the manipulated plots when compared with the respective unmanipulated treatments, indicating that shrub canopy cover was a dominant factor influencing the soil thermal regime. Conversely, we found no strong evidence of increased soil decomposition, CO2 fluxes, or nitrate or ammonia adsorbtion under artificial shrub canopy treatments when compared with unmanipulated open tundra. Our results suggest that the abiotic influences of shrub canopy cover alone on nutrient dynamics are weaker than previously asserted.


Chromosomal assignment of genes involved in glycosylphosphatidylinositol anchor biosynthesis: implications for the pathogenesis of paroxysmal nocturnal hemoglobinuria.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that affects both sexes equally. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all PNH patients tested to date, the biosynthetic defect occurs at the addition of N-acetyl-glucosamine to the phosphatidylinositol molecule (class A defect). A human cDNA, Piga, that repairs cell lines with the class A GPI-anchor biosynthetic defect has been recently cloned. Mapping of Piga to the X chromosome suggests that a single acquired mutation within Piga could alter GPI-anchor synthesis and result in PNH. However, this finding does not explain why all PNH patients have the class A defect. In the current study, the chromosomal assignment of Piga, as well as of Pigf and Pigh, two additional genes involved in GPI-anchor biosynthesis, has been established using a mouse interspecific backcross mapping technique. In contrast to Piga, both human and mouse Pigf and Pigh genes map to autosomes. The location of Pigf and Pigh suggests that mutations on both alleles of these autosomal genes would be necessary to produce PNH. This helps to explain the predominant class A defect in PNH.


Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9.
The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. Bacteria were recently found to have a form of adaptive immunity, the CRISPR-Cas systems, which use nucleic acid pairing to recognize and cleave genomic DNA of invaders such as bacteriophage. Historic work with tailed phages has shown that phage DNA is often modified by covalent attachment of large chemical groups. Here we demonstrate that DNA modification in phage T4 inhibits attack by the CRISPR-Cas9 system. This finding provides insight into mechanisms of host-virus competition and also a new set of tools that may be useful in modulating the activity of CRISPR-Cas9 in genome engineering applications.


Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples.
Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.


p90 ribosomal S6 kinase 3 contributes to cardiac insufficiency in α-tropomyosin Glu180Gly transgenic mice.
Myocardial interstitial fibrosis is an important contributor to the development of heart failure. Type 3 p90 ribosomal S6 kinase (RSK3) was recently shown to be required for concentric myocyte hypertrophy under in vivo pathological conditions. However, the role of RSK family members in myocardial fibrosis remains uninvestigated. Transgenic expression of α-tropomyosin containing a Glu180Gly mutation (TM180) in mice of a mixed C57BL/6:FVB/N background induces a cardiomyopathy characterized by a small left ventricle, interstitial fibrosis, and diminished systolic and diastolic function. Using this mouse model, we now show that RSK3 is required for the induction of interstitial fibrosis in vivo. TM180 transgenic mice were crossed to RSK3 constitutive knockout (RSK3(-/-)) mice. Although RSK3 knockout did not affect myocyte growth, the decreased cardiac function and mild pulmonary edema associated with the TM180 transgene were attenuated by RSK3 knockout. The improved cardiac function was consistent with reduced interstitial fibrosis in the TM180;RSK3(-/-) mice as shown by histology and gene expression analysis, including the decreased expression of collagens. The specific inhibition of RSK3 should be considered as a potential novel therapeutic strategy for improving cardiac function and the prevention of sudden cardiac death in diseases in which interstitial fibrosis contributes to the development of heart failure.


Effect of surface depressions on wetting and interactions between hydrophobic pore array surfaces.
The surface structure is known to significantly affect the long-range capillary forces between hydrophobic surfaces in aqueous solutions. It is, however, not clear how small depressions in the surface will affect the interaction. To clarify this, we have used the AFM colloidal probe technique to measure interactions between hydrophobic microstructured pore array surfaces and a hydrophobic colloidal probe. The pore array surfaces were designed to display two different pore spacings, 1.4 and 4.0 μm, each with four different pore depths ranging from 0.2 to 12.0 μm. Water contact angles measured on the pore array surfaces are lower than expected from the Cassie-Baxter and Wenzel models and not affected by the pore depth. This suggests that the position of the three-phase contact line, and not the interactions underneath the droplet, determines the contact angle. Confocal Raman microscopy was used to investigate whether water penetrates into the pores. This is of importance for capillary forces where both the movement of the three-phase contact line and the situation at the solid/liquid interface influence the stability of bridging cavities. By analyzing the shape of the force curves, we distinguish whether the cavity between the probe and the surfaces was formed on a flat part of the surface or in close proximity to a pore. The pore depth and pore spacing were both found to statistically influence the distance at which cavities form as surfaces approach each other and the distance at which cavities rupture during retraction.


Trained men show lower cortisol, heart rate and psychological responses to psychosocial stress compared with untrained men.
Physical activity has proven benefits for physical and psychological well-being and is associated with reduced responsiveness to physical stress. However, it is not clear to what extent physical activity also modulates the responsiveness to psychosocial stress. The purpose of this study was to evaluate whether the reduced responsiveness to physical stressors that has been observed in trained men can be generalized to the modulation of physiological and psychological responses to a psychosocial stressor. Twenty-two trained men (elite sportsmen) and 22 healthy untrained men were exposed to a standardized psychosocial laboratory stressor (Trier Social Stress Test). Adrenocortical (salivary free cortisol levels), autonomic (heart rate), and psychological responses (mood, calmness, anxiety) were repeatedly measured before and after stress exposure. In response to the stressor, cortisol levels and heart rate were significantly increased in both groups, without any baseline differences between groups. However, trained men exhibited significantly lower cortisol and heart rate responses to the stressor compared with untrained men. In addition, trained men showed significantly higher calmness and better mood, and a trend toward lower state anxiety during the stress protocol. On the whole, elite sportsmen showed reduced reactivity to the psychosocial stressor, characterized by lower adrenocortical, autonomic, and psychological stress responses. These results suggest that physical activity may provide a protective effect against stress-related disorders.


Conjunctival brush cytology.
In order to collect conjunctival cells efficiently, we developed a special brush that is a modification of the Cytobrush used in cervical cytology. The conjunctival brush is small and made of nylon bristles. Cells collected by these brushes were rinsed into a buffered solution, from which filter preparations were made. This technique produced adequately cellular samples from temporal bulbar conjunctiva; these preparations stained well with the Papanicolaou stain. Under normal conditions, three cell types were observed in the brushing samples; one was the polygonal epithelial cell, the second type was a small rounded cell, and the third type was a mucus-secreting goblet cell. Samples from dry-eyed patients contained keratinized cells with or without a decrease in goblet cells. Elongated cells were seen in samples from postirradiation and postoperative patients. Irritation caused by the brushing was of the same intensity as irritation caused by collecting cytologic specimens by impression or by the use of cotton swabs. These findings suggest that brushing cytology of the conjunctiva is a relatively noninvasive technique and can provide valuable information for evaluation of conjunctival conditions.


Multisystemic Therapy for social, emotional, and behavioral problems in youth aged 10-17.
Multisystemic Therapy (MST) is an intensive, home-based intervention for families of youth with social, emotional, and behavioral problems. Masters-level therapists engage family members in identifying and changing individual, family, and environmental factors thought to contribute to problem behavior. Intervention may include efforts to improve communication, parenting skills, peer relations, school performance, and social networks. Most MST trials were conducted by program developers in the USA; results of one independent trial are available and others are in progress. To provide unbiased estimates of the impacts of MST on restrictive out-of-home living arrangements, crime and delinquency, and other behavioral and psychosocial outcomes for youth and families. Electronic searches were made of bibliographic databases (including the Cochrane Library, C2-SPECTR, PsycINFO, Science Direct and Sociological Abstracts) as well as government and professional websites, from 1985 to January 2003. Reference lists of articles were examined, and experts were contacted. Studies where youth (age 10-17) with social, emotional, and/or behavioral problems were randomised to licensed MST programs or other conditions (usual services or alternative treatments). Two reviewers independently reviewed 266 titles and abstracts; 95 full-text reports were retrieved, and 35 unique studies were identified. Two reviewers independently read all study reports for inclusion. Eight studies were eligible for inclusion. Two reviewers independently assessed study quality and extracted data from these studies. Significant heterogeneity among studies was identified (assessed using Chi-square and I(2)), hence random effects models were used to pool data across studies. Odds ratios were used in analyses of dichotomous outcomes; standardised mean differences were used with continuous outcomes. Adjustments were made for small sample sizes (using Hedges g). Pooled estimates were weighted with inverse variance methods, and 95% confidence intervals were used. The most rigorous (intent-to-treat) analysis found no significant differences between MST and usual services in restrictive out-of-home placements and arrests or convictions. Pooled results that include studies with data of varying quality tend to favor MST, but these relative effects are not significantly different from zero. The study sample size is small and effects are not consistent across studies; hence, it is not clear whether MST has clinically significant advantages over other services. There is inconclusive evidence of the effectiveness of MST compared with other interventions with youth. There is no evidence that MST has harmful effects.


Shift Work and Poor Mental Health: A Meta-Analysis of Longitudinal Studies.
Background. Shift work is characterized by employees working outside the standard hours of 7:00 am to 6:00 pm. Because shift work includes night work, the normal sleep-wake cycle (circadian rhythm) is disrupted, with potential consequences for shift workers' physical and mental health.Objectives. To assess the pooled effects of shift work on mental health and to evaluate whether these differ in men and women.Search Methods. We searched PubMed, Scopus, and Web of Science databases for peer-reviewed or government reports published up to August 2018Selection Criteria. To be included, studies had to be longitudinal or case-control studies of shift work exposure associated with adverse mental health outcomes. For subanalyses, we grouped these outcomes as anxiety symptoms, depressive symptoms, or general poor mental health symptoms.Data Collection and Analysis. We followed the Meta-Analysis of Observational Studies in Epidemiology Group guidelines. We extracted adjusted risk estimates for each study to calculate pooled effect sizes (ESs) using random effect models and metaregression analysis to explore sources of heterogeneity.Main Results. We included 7 longitudinal studies, with 28 431 unique participants. Shift work was associated with increased overall risk of adverse mental health outcomes combined (ES = 1.28; 95% confidence interval [CI] = 1.02, 1.62; I2 = 70.6%) and specifically for depressive symptoms (ES = 1.33; 95% CI = 1.02, 1.74; I2 = 31.5%). Gender differences explained more than 90% of heterogeneity, with female shift workers more likely to experience depressive symptoms than female non-shift workers (odds ratio = 1.73; 95% CI = 1.39, 2.14).Authors' Conclusions. To our knowledge, this is the first meta-analysis to investigate the pooled effects of shift work on the risk of poor mental health, including subanalyses by type of poor mental health and gender. Shift workers, particularly women, are at increased risk for poor mental health, particularly depressive symptoms.Public Health Implications. Depression accounts for 4.3% of the global burden of disease and incidence, with mental disorders worldwide predicted to cost US $16.3 million by 2030. With 1 in 5 people in the United States and Europe doing shift work, and the increased risk of poor mental health among shift workers, shift work industries are a priority context for reducing this burden. Workplace health promotion programs and policies are needed to minimize shift workers' risk of poor mental health.


Gliomas and farm pesticide exposure in women: the Upper Midwest Health Study.
An excess incidence of brain cancer in male farmers has been noted in several studies, but few studies have focused on women. The National Institute for Occupational Safety and Health Upper Midwest Health Study evaluated effects of rural exposures for 341 female glioma cases and 528 controls, all adult (18-80 years of age) nonmetropolitan residents of Iowa, Michigan, Minnesota, and Wisconsin. On average, controls lived longer on farms than did cases. After adjusting for age, age group, education, and farm residence, no association with glioma was observed for exposure to arsenicals, benzoic acids, carbamates, chloroacetanilides, dinitroanilines, inorganics, organochlorines, organophosphates, phenoxys, triazines, or urea-based or estrogenic pesticides. An increased risk of glioma was observed for carbamate herbicides but was not statistically significant (odds ratio = 3.0; 95% confidence interval, 0.9-9.5). No association was observed between glioma and exposure to 12 widely used specific pesticides, after adjustment for age, age group, education, and any other pesticide exposure. These results were not affected after exclusion of proxy respondents (43% of cases, 2% of controls). Women were less likely than men to have applied pesticides, but more likely to have laundered pesticide-contaminated clothes. Storing pesticides in the house was associated with a statistically non-significant increased risk. Results show that exposure to pesticides was not associated with an increased risk of intracranial gliomas in women. Other farm-related factors could be etiologic factors and will be discussed in future reports.


A Telemonitoring and Hybrid Virtual Coaching Solution "CAir" for Patients with Chronic Obstructive Pulmonary Disease: Protocol for a Randomized Controlled Trial.
Chronic obstructive pulmonary disease (COPD) is one of the most common disorders in the world. COPD is characterized by airflow obstruction, which is not fully reversible. Patients usually experience breathing-related symptoms with periods of acute worsening and a substantial decrease in the health-related quality-of-life. Active and comprehensive disease management can slow down the progressive course of the disease and improve patients' disabilities. Technological progress and digitalization of medicine have the potential to make elaborate interventions easily accessible and applicable to a broad spectrum of patients with COPD without increasing the costs of the intervention. This study aims to develop a comprehensive telemonitoring and hybrid virtual coaching solution and to investigate its effects on the health-related quality of life of patients with COPD. A monocentric, assessor-blind, two-arm (intervention/control) randomized controlled trial will be performed. Participants randomized to the control group will receive usual care and a CAir Desk (custom-built home disease-monitoring device to telemonitor disease-relevant parameters) for 12 weeks, without feedback or scores of the telemonitoring efforts and virtual coaching. Participants randomized to the intervention group will receive a CAir Desk and a hybrid digital coaching intervention for 12 weeks. As a primary outcome, we will measure the delta in the health-related quality of life, which we will assess with the St. George Respiratory Questionnaire, from baseline to week 12 (the end of the intervention). The development of the CAir Desk and virtual coach has been completed. Recruitment to the trial started in September 2020. We expect to start data collection by December 2020 and expect it to last for approximately 18 months, as we follow a multiwave approach. We expect to complete data collection by mid-2022 and plan the dissemination of the results subsequently. To our knowledge, this is the first study investigating a combination of telemonitoring and hybrid virtual coaching in patients with COPD. We will investigate the effectiveness, efficacy, and usability of the proposed intervention and provide evidence to further develop app-based and chatbot-based disease monitoring and interventions in COPD. ClinicalTrials.gov identifier: NCT04373070; https://clinicaltrials.gov/ct2/show/NCT04373070. DERR1-10.2196/20412.


Novel Modifications of a Ventricular Assist Device for Infants and Children.
A continuous-flow "adult" ventricular assist device (VAD) was modified to support infants and children waiting for heart transplantation. A centrifugal VAD, designed to flow at 1.5 to 8 L/min, was used as a bridge to transplantation in pediatric patients. In smaller children and infants, a modified recirculation shunt permitted lower flow ranges. In hypoxic patients, an oxygenator was spliced into the circuit. From 2010 to 2015, the VAD was placed in 13 consecutive patients. Age ranged from 0.9 to 16 years (median, 7 years). Body surface area (BSA) ranged from 0.4 to 2.1 m(2) (median, 0.8 m(2)). Ten patients had a BSA less than 1.0 m(2). Four patients were receiving extracorporeal membrane oxygenation (ECMO) before VAD. Three patients had single-ventricle physiology. Five patients had a recirculation shunt and 3 underwent insertion of an oxygenator. Median time on the VAD was 20 days (range, 2-140 days). In patients with a recirculation shunt, mean patient flow was 1.5 L/min (mean flow/BSA, 2.7 L/min/m(2)), with mean total VAD flow of 3.4 L/min. Twelve patients underwent transplantation, and 1 patient underwent VAD explantation. All patients survived and were discharged at a median of 26 days (range, 17-83 days) after transplantation. Three patients experienced major bleeding events. There were 2 cerebrovascular accidents. VAD mortality dropped from 33% (3 of 9) during 2007 to 2010 to 0% (0 of 13) between 2011 and 2015 (p = 0.05). Wait-list mortality dropped from 10% (5 of 52) to 4% (4 of 91) for these periods (p = 0.29). The centrifugal VAD successfully supported pediatric patients awaiting heart transplantation. The modified recirculation shunt facilitated the successful support of patients in whom optimal flows were substantially lower than those recommended by the manufacturer. The design allows placement of an in-line oxygenator. Compared with pulsatile devices, use of this VAD was associated with a trend toward decreased mortality associated with VAD use.


Genetic evidence for difference between intracellular and extracellular peptides in influenza A matrix peptide-specific CTL recognition.
During the course of extensive mutagenesis of HLA-A2.1, we examined influenza A matrix peptide (FMP)-specific CTL recognition of HMy2.C1R (C1R) cells expressing mutant HLA-A2.1 molecules, sensitized with synthetic peptide, FMP 58-66, (exogenous peptide), or infected with influenza A virus (endogenous peptide). Most mutants showed equivalent presentation of exogenous and endogenous peptides to FMP-specific CTL. However, five of the mutants differed in this property. Two of the five mutants, F9L and T134K, present exogenous peptide to FMP-specific CTL, but fail to present endogenous peptide to CTL. Western blot analysis using anti-matrix protein Ab indicates that the matrix protein is expressed in these mutants after infection with virus. Interestingly, transfection of these two mutants with a minigene encoding FMP 58-66 results in efficient lysis by FMP-specific CTL. Peptide-binding assays demonstrate that the two mutations dramatically decrease the binding of FMP. However, these mutants bind FMP as well as wild type in the presence of exogenously added human beta 2-m, suggesting that the lower affinity for beta 2-m leads to the inability to present endogenous peptide. The remaining three mutants, Y27N, Q32K, and S132C, fail to present exogenous peptide, but present endogenous peptide to FMP-specific CTL. Pulse-chase analyses followed by endoglycosidase-H treatment show that the rate of maturation and processing of the five mutant HLA-A2 molecules in C1R cells is identical to that of wild type. Overall, this study suggests that the assembly and subsequent recognition of endogenous peptide differs from that of exogenous peptide.


Niraparib in patients with metastatic castration-resistant prostate cancer and DNA repair gene defects (GALAHAD): a multicentre, open-label, phase 2 trial.
Metastatic castration-resistant prostate cancers are enriched for DNA repair gene defects (DRDs) that can be susceptible to synthetic lethality through inhibition of PARP proteins. We evaluated the anti-tumour activity and safety of the PARP inhibitor niraparib in patients with metastatic castration-resistant prostate cancers and DRDs who progressed on previous treatment with an androgen signalling inhibitor and a taxane. In this multicentre, open-label, single-arm, phase 2 study, patients aged at least 18 years with histologically confirmed metastatic castration-resistant prostate cancer (mixed histology accepted, with the exception of the small cell pure phenotype) and DRDs (assessed in blood, tumour tissue, or saliva), with progression on a previous next-generation androgen signalling inhibitor and a taxane per Response Evaluation Criteria in Solid Tumors 1.1 or Prostate Cancer Working Group 3 criteria and an Eastern Cooperative Oncology Group performance status of 0-2, were eligible. Enrolled patients received niraparib 300 mg orally once daily until treatment discontinuation, death, or study termination. For the final study analysis, all patients who received at least one dose of study drug were included in the safety analysis population; patients with germline pathogenic or somatic biallelic pathogenic alterations in BRCA1 or BRCA2 (BRCA cohort) or biallelic alterations in other prespecified DRDs (non-BRCA cohort) were included in the efficacy analysis population. The primary endpoint was objective response rate in patients with BRCA alterations and measurable disease (measurable BRCA cohort). This study is registered with ClinicalTrials.gov, NCT02854436. Between Sept 28, 2016, and June 26, 2020, 289 patients were enrolled, of whom 182 (63%) had received three or more systemic therapies for prostate cancer. 223 (77%) of 289 patients were included in the overall efficacy analysis population, which included BRCA (n=142) and non-BRCA (n=81) cohorts. At final analysis, with a median follow-up of 10·0 months (IQR 6·6-13·3), the objective response rate in the measurable BRCA cohort (n=76) was 34·2% (95% CI 23·7-46·0). In the safety analysis population, the most common treatment-emergent adverse events of any grade were nausea (169 [58%] of 289), anaemia (156 [54%]), and vomiting (111 [38%]); the most common grade 3 or worse events were haematological (anaemia in 95 [33%] of 289; thrombocytopenia in 47 [16%]; and neutropenia in 28 [10%]). Of 134 (46%) of 289 patients with at least one serious treatment-emergent adverse event, the most common were also haematological (thrombocytopenia in 17 [6%] and anaemia in 13 [4%]). Two adverse events with fatal outcome (one patient with urosepsis in the BRCA cohort and one patient with sepsis in the non-BRCA cohort) were deemed possibly related to niraparib treatment. Niraparib is tolerable and shows anti-tumour activity in heavily pretreated patients with metastatic castration-resistant prostate cancer and DRDs, particularly in those with BRCA alterations. Janssen Research & Development.


Steroid hormone-dependent expression of blocker-sensitive ENaCs in apical membranes of A6 epithelia.
Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73-112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 microA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6-20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging approximately 0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.


Effect of single-dose ibuprofen on follicular fluid levels of interleukins in poor responders undergoing in vitro fertilization.
Premature follicular rupture during in vitro fertilization (IVF) is a well-known culprit for cycle cancellation. We sought to evaluate whether a single oral dose of ibuprofen will have an effect on the follicular fluid (FF) levels of inflammatory markers involved in ovulation. This is a prospective within-subjects study following nine patients undergoing IVF. Every patient underwent a first cycle of minimal stimulation IVF followed by a second cycle using the same stimulation protocol, except one oral dose of ibuprofen 800 mg was administered 15-18 h post-trigger injection. FF was obtained during oocyte retrievals of both cycles and analyzed for levels of selected inflammatory markers. A total of 27 cytokines and 9 matrix metalloproteinases (MMPs) were tested. Results demonstrate significantly decreased levels of interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor (G-CSF), eotaxin, MMP3, MMP7, MMP12, and MMP13 in FF of cycles where ibuprofen was administered. However, other cytokines levels, such IL-1 and vascular endothelial growth factor (VEGF), were similar with or without ibuprofen. Levels of MMPs described to be involved in ovulation, namely MMP-2 and MMP-9, were either undetectable or unchanged by ibuprofen, respectively. In conclusion, our data show that one dose of ibuprofen administered orally the day after trigger injection revealed a significant impact on the FF inflammatory milieu. Abbreviations: IVF: in vitro fertilization; MMP: matrix metalloproteinase; IL: interleukins; FF: follicular fluid; VEGF: vascular endothelial growth factor; NSAIDS: non-steroidal anti-inflammatories; POR: poor ovarian response; AMH: anti-Mullerian hormone; TAFC: total antral follicle count; HMG: human menopausal gonadotropin; hCG: human chorionic gonadotropin; COX: cyclooxygenase enzymes; PGH2: prostaglandin H2; RANTES: regulated on activation, normal T expressed and secreted; NF-κb: nuclear factor kappa-light-chain-enhancer of activated B cells.


MP-Link cementless distal fixation modular prosthesis for revision total hip arthroplasty.
The aseptic loosening of the femoral stem may require different treatments due to the complex problems related to the bone resorption (qualitatively and quantitatively): a minimum bone stock loss may result in a periprosthetic resorption that is so severe to make the fixation of the revision prosthesis extremely difficult. The revision surgery of a loosened hip prosthesis is often characterized by a complex reconstruction due to such severe loss of femoral bone tissue. We illustrate our experience with MP-Link modular stem (Waldemar Link, Hamburg, Germany) in cases of severe bone loss of the metaphyseal area and of the proximal third of the femoral shaft, assessing the technological developments (materials and design) over the years and examining the pros and cons of cementless distalfixation modular stems versus the traditional Wagner's stem.


DeepTracer-ID: De novo protein identification from cryo-EM maps.
The recent revolution in cryo-electron microscopy (cryo-EM) has made it possible to determine macromolecular structures directly from cell extracts. However, identifying the correct protein from the cryo-EM map is still challenging and often needs additional sequence information from other techniques, such as tandem mass spectrometry and/or bioinformatics. Here, we present DeepTracer-ID, a server-based approach to identify the candidate protein in a user-provided organism de novo from a cryo-EM map, without the need for additional information. Our method first uses DeepTracer to generate a protein backbone model that best represents the cryo-EM map, and this model is then searched against the library of AlphaFold2 predictions for all proteins in the given organism. This method is highly accurate and robust for high-resolution cryo-EM maps: in all 13 experimental maps tested blindly, DeepTracer-ID identified the correct proteins as the top candidates. Eight of the maps were of known structures, while the other five unpublished maps were validated by prior protein annotation and careful inspection of the model refined into the map. The program also showed promising results for both homomeric and heteromeric protein complexes. This platform is possible because of the recent breakthroughs in large-scale three-dimensional protein structure prediction.


Evaluation of role of Candida in patients with chronic paronychia.
Chronic paronychia, earlier considered to be an infection due to Candida, is currently being considered as a dermatitis of the nail fold. Irritant, allergic and protein contact dermatitis are the suggested major pathogenic mechanisms. Hypersensitivity to Candida is more likely to be the etiology, rather than the infection itself. To assess the clinico-etiological profiles of patients with chronic paronychia and to determine the role of contact sensitization and hypersensitivity to Candida. All consecutive patients of chronic paronychia attending the dermatology outpatient department (OPD) were assessed for risk factors, number of nails affected, clinical presentation and presence of fungus, patch tested for contact allergy and prick tested for hypersensitivity to Candida allergen. A total of 80 patients of chronic paronychia were recruited into our study. There was female preponderance (66 patients, 82.5%), with the most common group affected being housewives (47 patients, 58.8%). Frequent washing of hands (64 patients, 80%) was the most common risk factor. Fungal culture was positive in 56.1% (41 patients), the predominant species cultured was Candida albicans (15 patients, 36.5%). Patch testing with Indian standard series was positive in 27.1% patients (19 out of 70 patients tested), with nickel being the most common allergen. Prick test with Candida allergen was positive in 47.6% patients (31 out of 65 patients tested). Prick test and patch test provide indirect evidence of hypersensitivity, with inherent limitations. Our study shows that chronic paronychia is probably a form of hand dermatitis associated with prolonged wet work, and that there is a higher incidence of contact sensitization and Candida hypersensitivity in these patients.


Prematurity, sudden infant death syndrome, and age of death.
To determine if preterm infants are at greater risk for sudden infant death syndrome (SIDS) than term infants and to determine if the postconceptional age of SIDS deaths varies by gestational age at birth. A cohort analysis was conducted using data from the 1987 United States' Birth Cohort Linked Birth/Infant Death Certificate tapes. SIDS was defined as the death of any infant who was > 24 weeks gestation at birth; weighed > 500 g at birth; was assigned an International Classification of Diseases-9th Revision (ICD-9) underlying cause of death of 7980; and had an autopsy. The overall SIDS rate using our definition was 1.20 deaths/1000 live births. The SIDS rates by gestational age categories of 24 to 28 weeks, 29 to 32 weeks, 33 to 36 weeks, and 37 or more weeks were 3.52, 3.01, 2.27, and 1.06 deaths/1000 live births, respectively. Because of misclassification of gestational age among the most preterm infants, a restricted analysis was conducted on SIDS victims whose gestational ages fell within cutoff values derived from a methodology that excluded gestational age assessments assumed to be invalid. This subgroup analysis showed a mean (standard deviates) postconceptional age of death for SIDS for infants of 24 to 28 weeks, 29 to 32 weeks, and 33 to 36 weeks gestation to be 45.8 (8.3), 47.3 (8.6), and 48.0 (8.3) weeks, respectively, compared with 52.3 (8.5) weeks for term infants (ANOVA P = .0001). We infer from this analysis that preterm infants are at higher risk for SIDS than term infants, and that the postconceptional age of peak vulnerability for SIDS may differ by 4 to 6 weeks between preterm and term infants.


Azacitidine for the treatment of myelodysplastic syndrome.
The myelodysplastic syndromes (MDS) encompass a heterogeneous group of malignant hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias, frequent karyotypic abnormalities and significant risk for transformation to acute myeloid leukemia (AML). The prognosis of patients with intermediate- or high-risk MDS is very poor. This is due to the fact that standard therapeutic options are largely palliative. Neither autologous stem cell transplantation (SCT) nor chemotherapeutic regimens have been shown to prolong survival in patients with MDS. Allogeneic SCT, while potentially curative, is only available to a selected group of patients and is associated with high morbidity and mortality in elderly patients, which constitute the majority of patients with MDS. Hypermethylation of tumor-suppressor genes has been invoked as an important pathogenetic mechanism in MDS. The pyrimidine nucleoside analog azacitidine, which inhibits DNA methyltransferases, has recently become the first therapeutic to prolong survival in patients with MDS, thus changing the natural history of these malignancies. The activity of azacitidine in MDS has spurred the development of combinations of this agent with other epigenetic modifiers for the treatment of MDS and AML.


A placebo-controlled clinical trial of L-tryptophan in premenstrual dysphoria.
Antidepressant drugs, including specific serotonin reuptake inhibitors, have been shown to be beneficial in the treatment of premenstrual dysphoric disorders. The present study tests the efficacy of L-tryptophan, which acts specifically on serotonergic neurons, in this disorder. In a randomized controlled clinical trial, 37 patients with premenstrual dysphoric disorder were treated with L-tryptophan 6 g per day, and 34 were given placebo. The treatments were administered under double-blind conditions for 17 days, from the time of ovulation to the third day of menstruation, during three consecutive menstrual cycles. The Visual Analogue Scales (VAS) revealed a significant (p = .004) therapeutic effect of L-tryptophan relative to placebo for the cluster of mood symptoms comprising the items of dysphoria, mood swings, tension, and irritability. The magnitude of the reduction from baseline in maximum luteal phase VAS-mood scores was 34.5% with L-tryptophan compared to 10.4% with placebo. These results suggest that increasing serotonin synthesis during the late luteal phase of the menstrual cycle has a beneficial effect in patients with premenstrual dysphoric disorder.


Evaluation of in vitro gas production and rumen bacterial populations fermenting corn milling (co)products.
The objective of this study was to evaluate the fermentation dynamics of 2 commonly fed corn (co)products in their intact and defatted forms, using the in vitro gas production (IVGP) technique, and to investigate the shifts of the predominant rumen bacterial populations using the 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) technique. The bTEFAP technique was used to determine the bacterial profile of each fermentation time at 24 and 48 h. Bacterial populations were identified at the species level. Species were grouped by substrate affinities (guilds) for cellulose, hemicellulose, pectin, starch, sugars, protein, lipids, and lactate. The 2 (co)products were a dried distillers grain (DDG) plus solubles produced from a low-heat drying process (BPX) and a high-protein DDG without solubles (HP). Chemical analysis revealed that BPX contained about 11.4% ether extract, whereas HP contained only 3.88%. Previous studies have indicated that processing methods, as well as fat content, of corn (co)products directly affect fermentation rate and substrate availability, but little information is available regarding changes in rumen bacterial populations. Fermentation profiles of intact and defatted BPX and HP were compared with alfalfa hay as a standard profile. Defatting before incubation had no effect on total gas production in BPX or HP, but reduced lag time and the fractional rate of fermentation of BPX by at least half, whereas there was no effect for HP. The HP feed supported a greater percentage of fibrolytic and proteolytic bacteria than did BPX. Defatting both DDG increased the fibrolytic (26.8 to 38.7%) and proteolytic (26.1 to 37.2%) bacterial guild populations and decreased the lactate-utilizing bacterial guild (3.06 to 1.44%). Information regarding the fermentation kinetics and bacterial population shifts when feeding corn (co)products may lead to more innovative processing methods that improve feed quality (e.g., deoiling) and consequently allow greater inclusion rates in dairy cow rations.


Two-way vapochromism of a luminescent platinum(ii) complex with phosphonic-acid-functionalized bipyridine ligand.
A luminescent Pt(ii) complex [Pt(CN)2(H4dpbpy)] (1P; H4dpbpy = 2,2'-bipyridine-4,4'-diphosphonic acid) bearing a phosphonic-acid-functionalized bipyridine ligand was successfully synthesized and its unique two-way vapochromic behaviour investigated. X-ray structure analyses of both the anhydrous 1P and penta-hydrated 1P·5H2O phases clearly reveal the activation of intermolecular PtPt interactions through the adsorption of water vapour. Emission spectroscopy reveals that the penta-hydrated 1P·5H2O complex exhibits an orange emission at 585 nm that shifts in two directions, to a blue-green emission at 469 nm by drying at 100 °C or to a red emission at 701 nm by drying under vacuum at room temperature. Thermogravimetric analyses and powder X-ray diffraction studies clearly reveal that anhydrous 1P, with negligible intermolecular PtPt interactions, is formed by drying at 100 °C whereas the monohydrate 1P·H2O phase, with effective PtPt interactions, is formed by drying under vacuum.


Connecting the innate and adaptive immune responses in mouse choroidal neovascularization via the anaphylatoxin C5a and γδT-cells.
Neovascular age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). An overactive complement system is associated with AMD pathogenesis, and serum pro-inflammatory cytokines, including IL-17, are elevated in AMD patients. IL-17 is produced by complement C5a-receptor-expressing T-cells. In murine CNV, infiltrating γδT- rather than Th17-cells produce the IL-17 measurable in lesioned eyes. Here we asked whether C5a generated locally in response to CNV recruits IL-17-producing T-cells to the eye. CNV lesions were generated using laser photocoagulation and quantified by imaging; T-lymphocytes were characterized by QRT-PCR. CNV resulted in an increase in splenic IL-17-producing γδT- and Th17-cells; yet in the CNV eye, only elevated levels of γδT-cells were observed. Systemic administration of anti-C5- or anti-C5a-blocking antibodies blunted the CNV-induced production of splenic Th17- and γδT-cells, reduced CNV size and eliminated ocular γδT-cell infiltration. In ARPE-19 cell monolayers, IL-17 triggered a pro-inflammatory state; and splenocyte proliferation was elevated in response to ocular proteins. Thus, we demonstrated that CNV lesions trigger a systemic immune response, augmenting local ocular inflammation via the infiltration of IL-17-producing γδT-cells, which are presumably recruited to the eye in a C5a-dependent manner. Understanding the complexity of complement-mediated pathological mechanisms will aid in the development of an AMD treatment.


Biomechanical Effects of an Injury Prevention Program in Preadolescent Female Soccer Athletes.
Anterior cruciate ligament (ACL) injuries are common, and children as young as 10 years of age exhibit movement patterns associated with an ACL injury risk. Prevention programs have been shown to reduce injury rates, but the mechanisms behind these programs are largely unknown. Few studies have investigated biomechanical changes after injury prevention programs in children. Purpose/Hypothesis: To investigate the effects of the F-MARC 11+ injury prevention warm-up program on changes to biomechanical risk factors for an ACL injury in preadolescent female soccer players. We hypothesized that the primary ACL injury risk factor of peak knee valgus moment would improve after training. In addition, we explored other kinematic and kinetic variables associated with ACL injuries. Controlled laboratory study. A total of 51 female athletes aged 10 to 12 years were recruited from soccer clubs and were placed into an intervention group (n = 28; mean [±SD] age, 11.8 ± 0.8 years) and a control group (n = 23; mean age, 11.2 ± 0.6 years). The intervention group participated in 15 in-season sessions of the F-MARC 11+ program (2 times/wk). Pre- and postseason motion capture data were collected during preplanned cutting, unanticipated cutting, double-leg jump, and single-leg jump tasks. Lower extremity joint angles and moments were estimated using OpenSim, a biomechanical modeling system. Athletes in the intervention group reduced their peak knee valgus moment compared with the control group during the double-leg jump (mean [±standard error of the mean] pre- to posttest change, -0.57 ± 0.27 %BW×HT vs 0.25 ± 0.25 %BW×HT, respectively; P = .034). No significant differences in the change in peak knee valgus moment were found between the groups for any other activity; however, the intervention group displayed a significant pre- to posttest increase in peak knee valgus moment during unanticipated cutting ( P = .044). Additional analyses revealed an improvement in peak ankle eversion moment after training during preplanned cutting ( P = .015), unanticipated cutting ( P = .004), and the double-leg jump ( P = .016) compared with the control group. Other secondary risk factors did not significantly improve after training, although the peak knee valgus angle improved in the control group compared with the intervention group during unanticipated cutting ( P = .018). The F-MARC 11+ program may be effective in improving some risk factors for an ACL injury during a double-leg jump in preadolescent athletes, most notably by reducing peak knee valgus moment. This study provides motivation for enhancing injury prevention programs to produce improvement in other ACL risk factors, particularly during cutting and single-leg tasks.


An asthmatic child with allergic bronchopulmonary aspergillosis (ABPA).
Özdemir Ö. An asthmatic child with allergic bronchopulmonary aspergillosis (ABPA). Turk J Pediatr 2018; 60: 446-452. Allergic bronchopulmonary aspergillosis (ABPA) results from a TH2 hypersensitivity lung disease in response to bronchial colonization of Aspergillus fumigatus in the respiratory tract. ABPA is a very rare disease in the pediatric population; the prevalence is anticipated to be about 1-2% in adult asthma patients and 2-15% in cystic fibrosis patients. In pediatric asthma patients, only rare case reports on ABPA have been published. We present a case of a 12-year-old boy admitted to our hospital with worsening (mild persistent) asthma symptoms such as a recurrent cough, episodic wheezing, and dyspnea for the last two years. He was diagnosed with pneumonia several times and suspected of having tuberculosis during this time. Chest X-ray showed bilaterally perihilar opacities and computed tomography findings revealed central bronchiectasis. With the clinical and laboratory findings, the patient fulfilled 7/8 major diagnostic criteria for ABPA. Treatment with oral corticosteroids and itraconazole was effective. To the best of our knowledge, this is the first reported case of pediatric ABPA with previously documented asthma from Turkey.


Geometrically enhanced morphology-dependent resonances of a dielectric sphere.
The effect that geometrical resonances of orbiting internally reflecting rays have on the morphology-dependent resonances of microspheres is investigated heuristically and numerically using generalized Lorenz-Mie theory. Angularly resolved off-axis Gaussian beam elastic scattering spectra are presented. The results obtained show that the elastic scattering intensity of morphology-dependent resonances is noticeably enhanced in the vicinity of the geometrical resonance scattering angles.


Internal screw fixation compared with bipolar hemiarthroplasty for treatment of displaced femoral neck fractures in elderly patients.
Internal fixation and arthroplasty are the two main options for the treatment of displaced femoral neck fractures in the elderly. The optimal treatment remains controversial. Using data from the Norwegian Hip Fracture Register, we compared the results of hemiarthroplasty and internal screw fixation in displaced femoral neck fractures. Data from 4335 patients over seventy years of age who had internal fixation (1823 patients) or hemiarthroplasty (2512 patients) to treat a displaced femoral neck fracture were compared at a minimum follow-up interval of twelve months. One-year mortality, the number of reoperations, and patient self-assessment of pain, satisfaction, and quality of life at four and twelve months were analyzed. Subanalyses of patients with cognitive impairment and reduced walking ability were done. In the arthroplasty group, only contemporary bipolar prostheses were used and uncemented prostheses with modern stems and hydroxyapatite coating accounted for 20.8% (522) of the implants. There were no differences in one-year mortality (27% in the osteosynthesis group and 25% in the arthroplasty group; p = 0.76). There were 412 reoperations (22.6%) performed in the osteosynthesis group and seventy-two (2.9%) in the hemiarthroplasty group during the follow-up period. After twelve months, the osteosynthesis group reported more pain (mean score, 29.9 compared with 19.2), higher dissatisfaction with the operation result (mean score, 38.9 compared with 25.7), and a lower quality of life (mean score, 0.51 compared with 0.60) than the arthroplasty group. All differences were significant (p < 0.001). For patients with cognitive impairment, hemiarthroplasty provided a better functional outcome (less pain, higher satisfaction with the result of the operation, and higher quality of life as measured on the EuroQol visual analog scale) at twelve months (p < 0.05). Displaced femoral neck fractures in the elderly should be treated with hemiarthroplasty.


Differential activation of eMI by distinct forms of cellular stress.
As one of the major, highly conserved catabolic pathways, autophagy delivers cytosolic components to lysosomes for degradation. It is essential for development, cellular homeostasis, and coping with stress. Reduced autophagy increases susceptibility to protein aggregation diseases and leads to phenotypes associated with aging. Of the three major forms of autophagy, macroautophagy (MA) can degrade organelles or aggregated proteins, and chaperone-mediated autophagy is specific for soluble proteins containing KFERQ-related targeting motifs. During endosomal microautophagy (eMI), cytoplasmic proteins are engulfed into late endosomes in an ESCRT machinery-dependent manner. eMI can be KFERQ-specific or occur in bulk and be induced by prolonged starvation. Its physiological regulation and function, however, are not understood. Here, we show that eMI in the Drosophila fat body, akin to the mammalian liver, is induced upon oxidative or genotoxic stress in an ESCRT and partially Hsc70-4-dependent manner. Interestingly, eMI activation is selective, as ER stress fails to elicit a response. Intriguingly, we find that reducing MA leads to a compensatory enhancement of eMI, suggesting a tight interplay between these degradative processes. Furthermore, we show that mutations in DNA damage response genes are sufficient to trigger eMI and that the response to oxidative stress is under the control of MAPK/JNK signaling. Our data suggest that, controlled by various signaling pathways, eMI allows an organ to react and adapt to specific types of stress and is thus likely critical to prevent disease.Abbreviations:Atg: autophagy-related; CMA: chaperone-mediated autophagy; DDR: DNA damage repair; Df: deficiency (deletion); (E)GFP: (enhanced) green fluorescent protein; eMI: endosomal microautophagy; ER: endoplasmatic reticulum; ESCRT: endosomal sorting complexes required for transport; Eto: etoposide; FLP: flipase; Hsc: heat shock cognate protein; LAMP2A: lysosomal-associated membrane protein 2A; LE: late endosome; MA: macroautophagy; MI: microautophagy; MVB: multivesicular body; PA: photoactivatable; Para: paraquat; ROS: reactive oxygen species; SEM: standard error of means; Tor: target of rapamycin [serine/threonine kinase]; UPR: unfolded protein response; Vps: vacuolar protein sorting.


Mining gene-disease relationships from biomedical literature: weighting protein-protein interactions and connectivity measures.
The promises of the post-genome era disease-related discoveries and advances have yet to be fully realized, with many opportunities for discovery hiding in the millions of biomedical papers published since. Public databases give access to data extracted from the literature by teams of experts, but their coverage is often limited and lags behind recent discoveries. We present a computational method that combines data extracted from the literature with data from curated sources in order to uncover possible gene-disease relationships that are not directly stated or were missed by the initial mining. An initial set of genes and proteins is obtained from gene-disease relationships extracted from PubMed abstracts using natural language processing. Interactions involving the corresponding proteins are similarly extracted and integrated with interactions from curated databases (such as BIND and DIP), assigning a confidence measure to each interaction depending on its source. The augmented list of genes and gene products is then ranked combining two scores: one that reflects the strength of the relationship with the initial set of genes and incorporates user-defined weights and another that reflects the importance of the gene in maintaining the connectivity of the network. We applied the method to atherosclerosis to assess its effectiveness. Top-ranked proteins from the method are related to atherosclerosis with accuracy between 0.85 to 1.00 for the top 20 and 0.64 to 0.80 for the top 90 if duplicates are ignored, with 45% of the top 20 and 75% of the top 90 derived by the method, not extracted from text. Thus, though the initial gene set and interactions were automatically extracted from text (and subject to the impreciseness of automatic extraction), their use for further hypothesis generation is valuable given adequate computational analysis.


WearSense: Detecting Autism Stereotypic Behaviors through Smartwatches.
Autism is a complex developmental disorder that affects approximately 1 in 68 children (according to the recent survey conducted by the Centers for Disease Control and Prevention-CDC) in the U.S., and has become the fastest growing category of special education. Each student with autism comes with her or his own unique needs and an array of behaviors and habits that can be severe and which interfere with everyday tasks. Autism is associated with intellectual disability, impairments in social skills, and physical health issues such as sleep and abdominal disturbances. We have designed an Internet-of-Things (IoT) framework named WearSense that leverages the sensing capabilities of modern smartwatches to detect stereotypic behaviors in children with autism. In this work, we present a study that used the inbuilt accelerometer of a smartwatch to detect three behaviors, including hand flapping, painting, and sibbing that are commonly observed in children with autism. In this feasibility study, we recruited 14 subjects to record the accelerometer data from the smartwatch worn on the wrist. The processing part extracts 34 different features in each dimension of the three-axis accelerometer, resulting in 102 features. Using and comparing various classification techniques revealed that an ensemble of 40 decision trees has the best accuracy of around 94.6%. This accuracy shows the quality of the data collected from the smartwatch and feature extraction methods used in this study. The recognition of these behaviors by using a smartwatch would be helpful in monitoring individuals with autistic behaviors, since the smartwatch can send the data to the cloud for comprehensive analysis and also to help parents, caregivers, and clinicians make informed decisions.


Neutrophil-Derived Proteases in the Microenvironment of Pancreatic Cancer -Active Players in Tumor Progression.
A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the fibro-inflammatory microenvironment, consisting of activated pancreatic stellate cells, extracellular matrix proteins, and a variety of inflammatory cells, such as T cells, macrophages, or neutrophils. Tumor-infiltrating immune cells, which are found in nearly all cancers, including PDAC, often fail to eliminate the tumor, but conversely can promote its progression by altering the tumor microenvironment. Pancreatic cancer cells are able to attract polymorphonuclear neutrophils (PMN) via tumor secreted chemokines and in human PDAC, PMN infiltrates can be observed in the vicinity of tumor cells and in the desmoplastic tumor stroma, which correlate with undifferentiated tumor growth and poor prognosis. The behavior of tumor-infiltrating neutrophils in the tumor micromilieu is not yet understood at a mechanistic level. It has been shown that PMN have the potential to kill tumor cells, either directly or by antibody-dependent cell-mediated cytotoxicity, but on the other side various adverse effects of PMN, such as promotion of aggressive tumor growth with epithelial-to-mesenchymal transition and increased metastatic potential, have been described. Recent therapeutic approaches for PDAC focus not only the tumor cell itself, but also elements of the tumor microenvironment. Therefore, the role of PMN and their derived products (e.g. cytokines, proteases) as a new vein for a therapeutic target should be critically evaluated in this context. This review summarizes the current understanding of the interplay between proteases of tumor-infiltrating neutrophils and pancreatic tumor cells and elements of the desmoplastic stroma.


Biomolecular corona formation: nature and bactericidal impact on surface-modified silica nanoparticles.
Biological fluids contain a plethora of different components that can coat nanoparticle's surface and critically affect their interaction with living systems. The recent literature has focused efforts on understanding the overall protein role in this nanoparticle's coating (commonly called a protein corona) without considering other components' influence. Here, we report a protocol to study the relationship between biological fluid components excluding proteins (referred to as a biomolecular corona) and nanoparticles. For this purpose, functionalized silica nanoparticles were duly synthesized to experimentally investigate the extent of biomolecular corona adsorption over different surfaces. This adsorption was directly correlated with the silica surface chemical constitution, while the extent of biomolecular corona coating affected the overall surface charge and the colloidal stability of the functionalized nanoparticles. In addition, the bactericidal properties of biomolecular corona-coated nanoparticles were tested against two medically relevant bacteria (Escherichia coli and Staphylococcus aureus). Biological results indicated that the presence and the extent of the biomolecular corona affect the bactericidal properties of the nanoparticles.


The effect of sequence context on the activity of cytosine DNA glycosylases.
We have prepared single (N204D) and double (N204D:L272A) mutants of human uracil DNA glycosylase (hUDG), generating two cytosine DNA glycosylases (hCDG and hCYDG). Both these enzymes are able to excise cytosine (but not 5-methylcytosine), when this base is part of a mismatched base pair. hCDG is more active than the equivalent E. coli enzyme (eCYDG) and also has some activity when the cytosine is paired with guanine, unlike eCYDG. hCDG also has some activity against single stranded DNA, while having poor activity towards an unnatural base pair that forces the cytosine into an extrahelical conformation (in contrast to eCYDG for which a bulky base enhances the enzyme's activity). We also examined how sequence context affects the activity of these enzymes, determining the effect of flanking base pairs on cleavage efficiency. An abasic site or a hexaethylene glycol linker placed opposite the target cytosine, also causes an increase in activity compared with an AC mismatch. Flanking an AC mismatch with GC base pairs resulted in a 100-fold decrease in excision activity relative to flanking AT base pairs and the 5'-flanking base pair had a greater effect on the rate of cleavage. However, this effect is not simply due to the stability of the flanking base pairs as adjacent GT mismatches also produce low cleavage efficiency.


Chronic assessment of cerebral hemodynamics during rat forepaw electrical stimulation using functional ultrasound imaging.
Functional ultrasound imaging is a method recently developed to assess brain activity via hemodynamics in rodents. Doppler ultrasound signals allow the measurement of cerebral blood volume (CBV) and red blood cells' (RBCs') velocity in small vessels. However, this technique originally requires performing a large craniotomy that limits its use to acute experiments only. Moreover, a detailed description of the hemodynamic changes that underlie functional ultrasound imaging has not been described but is essential for a better interpretation of neuroimaging data. To overcome the limitation of the craniotomy, we developed a dedicated thinned skull surgery for chronic imaging. This procedure did not induce brain inflammation nor neuronal death as confirmed by immunostaining. We successfully acquired both high-resolution images of the microvasculature and functional movies of the brain hemodynamics on the same animal at 0, 2, and 7 days without loss of quality. Then, we investigated the spatiotemporal evolution of the CBV hemodynamic response function (HRF) in response to sensory-evoked electrical stimulus (1 mA) ranging from 1 (200 μs) to 25 pulses (5s). Our results indicate that CBV HRF parameters such as the peak amplitude, the time to peak, the full width at half-maximum and the spatial extent of the activated area increase with stimulus duration. Functional ultrasound imaging was sensitive enough to detect hemodynamic responses evoked by only a single pulse stimulus. We also observed that the RBC velocity during activation could be separated in two distinct speed ranges with the fastest velocities located in the upper part of the cortex and slower velocities in deeper layers. For the first time, functional ultrasound imaging demonstrates its potential to image brain activity chronically in small animals and offers new insights into the spatiotemporal evolution of cerebral hemodynamics.


Anionic tobacco peroxidase is active at extremely low pH: veratryl alcohol oxidation with a pH optimum of 1.8.
Tobacco peroxidase (36 kDa, pI 3.5) exhibits unique catalytic and spectral properties that are modulated by pH, calcium and magnesium ions. It catalyses the oxidation of veratryl alcohol by hydrogen peroxide over a wide pH range (1.5-5.0) in the presence of these metal ions with a pH optimum of 1.8. This is the only example of a holoperoxidase described so far that is active and comparatively stable at such a low pH. The enhancement of tobacco peroxidase activity by magnesium ions is to our knowledge the first example of a magnesium-induced peroxidase activation. UV/visible spectra of tobacco peroxidase showed that the Soret band shifted and its absorption coefficient increased upon the addition of calcium or magnesium ions and on lowering the pH. The tobacco peroxidase spectrum at pH 1.85, in the presence of calcium chloride (> 50 mM), is similar to that of lignin peroxidase at pH 6.0, with the Soret band shifting from 403 to 409 nm and the molar absorption coefficient increasing from 108,000 to 148,000 +/- 2000 M-1.cm-1 (results given +/- S.E.M.; n = 3). The data provide evidence for a low-affinity site for bivalent metal ion binding in addition to the two constitutive calcium sites that are present in all plant peroxidases. The presence of a glutamic acid residue (Glu-141) at the entrance to the haem-binding pocket, analogous to Glu-146 in lignin peroxidase and not present in other plant peroxidases, may account for these novel properties.


A clinical study to optimise a sand fly biting protocol for use in a controlled human infection model of cutaneous leishmaniasis (the FLYBITE study).
Background: Leishmaniasis is a globally important yet neglected parasitic disease transmitted by phlebotomine sand flies. With new candidate vaccines in or near the clinic, a controlled human challenge model (CHIM) using natural sand fly challenge would provide a method for early evaluation of prophylactic efficacy. Methods : We evaluated the biting frequency and adverse effects resulting from exposure of human volunteers to bites of either Phlebotomus papatasi or P. duboscqi, two natural vectors of Leishmania major. 12 healthy participants were recruited (mean age 40.2 ± 11.8 years) with no history of significant travel to regions where L. major-transmitting sand flies are prevalent. Participants were assigned to either vector by 1:1 allocation and exposed to five female sand flies for 30 minutes in a custom biting chamber. Bite frequency was recorded to confirm a bloodmeal was taken. Participant responses and safety outcomes were monitored using a visual analogue scale (VAS), clinical examination, and blood biochemistry. Focus groups were subsequently conducted to explore participant acceptability. Results: All participants had at least one successful sand fly bite with none reporting any serious adverse events, with median VAS scores of 0-1/10 out to day 21 post-sand fly bite. Corresponding assessment of sand flies confirmed that for each participant at least 1/5 sand flies had successfully taken a bloodmeal (overall mean 3.67±1.03 bites per participant). There was no significant difference between P. papatasi and P. duboscqi in the number of bites resulting from 5 sand flies applied to human participants (3.3±0.81 vs 3.00±1.27 bites per participant; p=0.56) . In the two focus groups (n=5 per group), themes relating to positive participant-reported experiences of being bitten and the overall study, were identified. Conclusions: These results validate a protocol for achieving successful sand fly bites in humans that is safe, well-tolerated and acceptable for participants. Clinicaltrials.gov registration: NCT03999970 (27/06/2019).