Evaluating functional decline in patients with Multiple Sclerosis.
Multiple Sclerosis (MS) is a disease with a wide-ranging impact on functional status. The aim of the study was to examine the added value of simultaneously evaluating fatigue, personal ADL and handwriting performance as indicators for functional decline among patients with MS. Participants were 50 outpatients with MS and 26 matched healthy controls. Data collection instruments included a disability status scale, the Fatigue Severity Scale (FSS), and the Physical Self-Maintenance Scale (PSMS). Handwriting performance was evaluated by objective computerized measures of the handwriting process (ComPET). Significant differences were found between patients with MS and control subjects in their fatigue level, their PSMS score and In-air time per stroke while writing. The FSS together with specific PSMS items and handwriting measures achieved correct classification of 87.7% of the participants. These results are the first step towards demonstrating the added value of evaluating body function outcomes (fatigue) together with activity performance (handwriting and ADL) to document functional decline among patients with MS. These results may contribute to the development of practical intervention strategies targeted at improving performance abilities among patients with MS.


shRNA against CD44 inhibits cell proliferation, invasion and migration, and promotes apoptosis of colon carcinoma cells.
CD44 is a causal factor for tumor invasion, metastasis and acquisition of resistance to apoptosis. CD44 knockdown using inducible short hairpin RNA (shRNA) significantly reduces cell growth and invasion. Short hairpin RNA against CD44 and pGFP-V-RS-vector was used for knockdown of CD44 expression in SW620 colon cancer cells. Cell growth, invasion and migration assay, immunofluorescence for β-catenin expression and western blotting for Wnt signaling molecules were analyzed. Cell cycle analysis and western blot analysis for apoptotic molecules were evaluated. Short hairpin RNA against CD44 reduced the expression of CD44. Cell proliferation, migration and invasion were markedly inhibited and apoptosis was increased in shRNA CD44-transfected cells. Knockdown of CD44 decreased the phosphorylation of PDK1, Akt and GSK3β, and β-catenin levels. Decreased phosphorylated Akt led to an increase in phosphorylated FoxO1 and induced cell cycle arrest in the G0-G1 phase and a decrease in the S phase. The levels of Bcl-2 and Bcl-xL expression were down-regulated, while the levels of BAX expression and cleaved caspase-3, -8 and -9 were increased. CD44 knockdown by way of shRNA inhibited cell proliferation and induced cell apoptosis. This can be used as a therapeutic intervention with the anti-survival/pro-apoptotic machinery in human colon cancer.


[Effect of Thrombocytopenia on Migration and Homing Pattern of Dendritic Cells].
To investigate the effect of thrombocytopenia on the migration patterns of adoptive dendritic cell(DC) in vivo. The mouse model of thrombocytopenia was established by intraperitoneal administration of anti-CD41 mAb MWReg30. Mouse bone marrow(BM)-derived DC were injected into thrombocytopenia mouse by footpad infusion and intravenous infusion. The DC migration and distribution pattern were detected by bioluminescence imaging. More than 80% platelets were cleared in the experimental group which was infused with anti-CD41 antibody. At 72 h after injection, the percentage of injected DC that migrated from footpad to popliteal lymph nodes(PLNs) and inguinal lymph nodes(ILNs) were (0.32±0.02)% and (0.02±0.01)% in experimental group, and (0.27±0.15)% and (0.02±0.02)% in control group, respectively. Statistic data showed that there was no statistical difference between these 2 groups (P>0.05). The issue distribution pattern of intravenously injected DC between experimental group and control group were not distinctly different, and large amounts of injected DC accumulated in the spleen, liver draining lymph-nodes lungs and liver. Thrombocytopenia has not a distinct effect on the migratory capacity and tissue distribution of DC by either footpad or intravenous injection.


Cholesterol and coronary heart disease. The 21st century.
Recent clinical trials have demonstrated that reductions of serum low-density lipoprotein (LDL) levels substantially decrease the risk for coronary heart disease. These trials confirm other lines of evidence that high levels of LDL are a critical atherogenic factor. Aggressive lowering of LDL levels in high-risk patients promises to significantly reduce morbidity and mortality from coronary heart disease in the first third of the 21st century. However, several additional measures will be required to marginalize coronary heart disease in the 21st century. Other lipoprotein abnormalities and other risk factors, eg, cigarette smoking, hypertension, and diabetes mellitus, must be controlled to obtain the full benefit of LDL-lowering therapy. Moreover, the health care delivery system must be reorganized to put more emphasis on prevention. Although much can be achieved through application of current knowledge in prevention efforts, further advances through new research will be required to remove coronary heart disease as a major cause of death in the United States.


Three-dimensional reconstruction of collagen-proteoglycan interactions in the mouse corneal stroma by electron tomography.
Corneal transparency is fundamental to the visual system, and is directly related to the ordered collagen fibril architecture that the cornea maintains. Proteoglycans, through their protein core and highly anionic glycosaminoglycan side chains, are thought to regulate the collagen organisation in the corneal stroma. To understand the inter-relationships between proteoglycans and collagen fibrils in the cornea, adult mouse corneas were treated with cuprolinic blue and three-dimensional reconstructions of the anterior, mid and posterior corneal stroma were obtained. The reconstructions show regular diameters of collagen fibrils throughout the cornea and uniform interfibrillar spacing within each region. Both longitudinal and transverse reconstructions were obtained to establish a clear picture of proteoglycan organisation, yet no distinct regular pattern or symmetry of proteoglycan orientation was observed. Large, electron-dense proteoglycans (possibly chondroitin sulphate/dermatan sulphate proteoglycans) interconnecting two or often three adjacent collagen fibrils are seen, whilst another sub-population of smaller proteoglycans (of the keratan sulphate variety) interconnect only neighbouring fibrils. The reconstructions suggest a complex interaction between proteoglycans and collagen, which allows for the dynamic control of collagen fibril architecture in the cornea.


Mosquito-borne viruses and suppressors of invertebrate antiviral RNA silencing.
The natural maintenance cycles of many mosquito-borne viruses require establishment of persistent non-lethal infections in the invertebrate host. While the mechanisms by which this occurs are not well understood, antiviral responses directed by small RNAs are important in modulating the pathogenesis of viral infections in disease vector mosquitoes. In yet another example of an evolutionary arms race between host and pathogen, some plant and insect viruses have evolved to encode suppressors of RNA silencing (VSRs). Whether or not mosquito-borne viral pathogens encode VSRs has been the subject of debate. While at first there would seem to be little evolutionary benefit to mosquito-borne viruses encoding proteins or sequences that strongly interfere with RNA silencing, we present here a model explaining how the expression of VSRs by these viruses in the vector might be compatible with the establishment of persistence. We also discuss the challenges associated with interrogating these viruses for the presence of suppressor proteins or sequences, as well as the candidates that have been identified in the genomes of mosquito-borne pathogens thus far.


Measurement and mitigation of free convection in microfluidic gradient generators.
Microfluidic gradient generators are used to study the movement of living cells, lipid vesicles, and colloidal particles in response to spatial variations in their local chemical environment. Such gradient driven motions are often slow (less than 1 μm s-1) and therefore influenced or disrupted by fluid flows accompanying the formation and maintenance of the applied gradient. Even when external flows are carefully eliminated, the solute gradient itself can drive fluid motions due to combinations of gravitational body forces and diffusioosmotic surface forces. Here, we develop a microfluid gradient generator based on the in situ formation of biopolymer membranes and quantify the fluid flows induced by steady solute gradients. The measured velocity profiles agree quantitatively with those predicted by analytical approximations of relevant hydrodynamic models. We discuss how the speed of gradient-driven flows depends on system parameters such as the gradient magnitude, the fluid viscosity, the channel dimensions, and the solute type. These results are useful in identifying and mitigating undesired flows within microfluidic gradient systems.


A major species of mouse mu-opioid receptor mRNA and its promoter-dependent functional polyadenylation signal.
The pharmacological effects of opioid drugs are mediated mainly by the mu-opioid receptor (MOR), which is encoded by an mRNA transcript named MOR1. Although several MOR mRNA splice variants have been reported, their biological relevance has been debated. In this study, we found that probes of regions essential for the production of functional MOR, as well as that of the 3'-downstream region of the MOR gene coding region, detected by Northern blot analyses, a major species of mature transcript MOR1 from mouse brain of approximately 11.5 kilobases (kb). Although exon 3 probe detected an additional 3.7-kb transcript, this transcript was not detected by other probes, ruling out its ability to produce functional MOR. The 3'-untranslated region (UTR) of MOR1 is contiguously extended from the end of the coding region, and uses a single polyadenylation [poly (A)] signal (located 10,179 bp downstream of the MOR1 stop codon). The poly (A) signal (AAUAAA) is located 26 bp upstream of the poly (A) site. Transient transfection using luciferase reporters verified the functionality of this poly (A) signal, in particular on a reporter driven by the MOR promoter. This poly (A) is much less effective for a heterologous promoter, such as simian virus 40, indicating a functional coupling of MOR promoter and its own poly (A). This report verifies MOR1 as the major mature MOR gene transcript that has the full capacity to produce functional MOR protein, identifies the 3'-UTR of MOR1 transcript, and uncovers functional coupling of the MOR gene promoter and its polyadenylation signal.


Lipid peroxidation in patients with brain tumor.
Molecular and genetic signatures may predict brain tumor behavior and may soon guide tumor classification, diagnosis, and tumor-specific treatment strategies. Free oxygen radicals (FOR) are thought to take part in oncogenesis and cellular differentiation. This article explored the state of FORs and antioxidant system in patients with cerebral tumor. The serum concentrations of malondialdehyde (MDA), catalase, and glutathione peroxidase (GSH-Px) enzyme activities were measured in the serum of 35 patients with cerebral tumors (21 glioma, 14 meningioma) and 11 controls. MDA measurement was done with fluorometric method and catalase and GSH-Px enzyme activities were done with photometric method. Mean serum MDA levels, catalase, and GSH-Px enzyme activities were significantly higher for both glial and meningiomal tumor cases when compared to controls (p < .05). There is no significant difference between glioma and meningioma groups in terms of the aforementioned parameters (p > .05). In conclusion, lipid peroxidation and antioxidant enzymes as assessed by MDA, catalase, and GSH-Px were increased in patients with brain tumors, for this respect there is no difference between gliomas and meningiomas.


Investigation on changes in complex vegetation coverage using multi-temporal landsat data of Western Black Sea region--a case study.
Previous studies have been able to successfully detect changes in gently-sloping forested areas with low-diversity and homogeneous vegetation cover using medium-resolution satellite data such as landsat. The aim of the present study is to examine the capacity of multi-temporal landsat data to identify changes in forested areas with mixed vegetation and generally located on steep slopes or non-uniform topography landsat thematic mapper (TM) and landsat enhanced thematic mapperplus (ETM+) data for the years 1987-2000 was used to detect changes within a 19,500 ha forested area in the Western Black sea region of Turkey. The data comply with the forest cover type maps previously created for forest management plans of the research area. The methods used to detect changes were: post-classification comparison, image differencing, image rationing and NDVI (Normalized Difference Vegetation Index) differencing methods. Following the supervised classification process, error matrices were used to evaluate the accuracy of classified images obtained. The overall accuracy has been calculated as 87.59% for 1987 image and as 91.81% for 2000 image. General kappa statistics have been calculated as 0.8543 and 0.9038 for 1987 and 2000, respectively. The changes identified via the post-classification comparison method were compared with other change detetion methods. Maximum coherence was found to be 74.95% at 4/3 band rate. The NDVI difference and 3rd band difference methods achieved the same coherence with slight variations. The results suggest that landsat satellite data accurately conveys the temporal changes which occur on steeply-sloping forested areas with a mixed structure, providing a limited amount of detail but with a high level of accuracy. Moreover it has been decided that the post-classification comparison method can meet the needs of forestry activities better than other methods as it provides information about the direction of these changes.


Cytokines, inducers and inhibitors modulate MMP-2 and MMP‑9 secretion by human Fanconi anemia immortalized fibroblasts.
Acute myeloid leukemia and head and neck squamous cell carcinomas are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated in tumor invasion and metastasis. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human immortalized fibroblasts from FA. Human FA immortalized fibroblast cell lines FA-A:PD220 and FA-D2:PD20 were grown in minimum essential medium (MEM) supplemented with 15% fetal bovine serum (FBS) and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with phosphate‑buffered saline (PBS) and incubated in serum-free media with the following: phorbol 12-myristate 13-acetate (PMA) at 10-100 ng/ml; tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) at 0.1-25 ng/ml; lipopolysaccharide (LPS) at 10-100 µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10-100 µM without and with PMA; a nutrient mixture (NM) without and with PMA at 10-1,000 µg/ml; actinomycin-D and cyclohexamide at 2 and 4 µM; retinoic acid and dexamethasone at 50 µM. After 24 h, media were removed and analyzed for MMP-2 and MMP-9 by zymography. Both FA cell lines expressed only MMP-2 and responded similarly to cytokines, mitogens, inducers and inhibitors. PMA potently stimulated MMP-9 and had a moderate effect on MMP-2. TNF-α showed variable effects on MMP-2 and significantly enhanced MMP-9. IL-1β enhanced MMP-2 slightly and MMP-9 significantly. LPS had a moderate stimulatory effect on MMP-2 and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated MMP-2 and MMP-9 expression. Actinomycin-D, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the NM and its component EGCG in the management of FA cancers.


De-identification procedures for magnetic resonance images and the impact on structural brain measures at different ages.
Surface rendering of MRI brain scans may lead to identification of the participant through facial characteristics. In this study, we evaluate three methods that overwrite voxels containing privacy-sensitive information: Face Masking, FreeSurfer defacing, and FSL defacing. We included structural T1-weighted MRI scans of children, young adults and older adults. For the young adults, test-retest data were included with a 1-week interval. The effects of the de-identification methods were quantified using different statistics to capture random variation and systematic noise in measures obtained through the FreeSurfer processing pipeline. Face Masking and FSL defacing impacted brain voxels in some scans especially in younger participants. FreeSurfer defacing left brain tissue intact in all cases. FSL defacing and FreeSurfer defacing preserved identifiable characteristics around the eyes or mouth in some scans. For all de-identification methods regional brain measures of subcortical volume, cortical volume, cortical surface area, and cortical thickness were on average highly replicable when derived from original versus de-identified scans with average regional correlations >.90 for children, young adults, and older adults. Small systematic biases were found that incidentally resulted in significantly different brain measures after de-identification, depending on the studied subsample, de-identification method, and brain metric. In young adults, test-retest intraclass correlation coefficients (ICCs) were comparable for original scans and de-identified scans with average regional ICCs >.90 for (sub)cortical volume and cortical surface area and ICCs >.80 for cortical thickness. We conclude that apparent visual differences between de-identification methods minimally impact reliability of brain measures, although small systematic biases can occur.


Lateral Flow Assay for Interleukin 6: A Technological and Scientific Prospection of a 10-Year Survey.
Interleukin-6 (IL-6) is a multifunctional cytokine that plays a crucial role not only in the immune system but also in numerous biological processes. Elevated IL-6 levels have been observed in many types of cancer and inflammatory diseases. Among the techniques that have been used to quantify IL-6, a Lateral Flow Assay (LFA) is one of the most promising. The objective of this study was to perform a technological and scientific exploration of the development of an LFA for IL-6. The Instituto Nacional de Propriedade Industrial-INPI (Brazil), European Patent Office - Espacenet, and United States Patent and Trademark Office-USPTO were used for the technological prospection to consider all the patents regarding an LFA for IL-6 detection. For the scientific investigation, the following periodic databases were used: Web of Science, Science Direct, PubMed, and Scielo, and were monitored from 2007 to May 2017. We found no patents based on the searched keywords, and a limited number of scientific articles were found. Therefore, the development of an LFA sensor for the detection of IL-6 appears to be innovative with significant biotechnological importance, creating a technology transfer to promote economic and industrial growth. The development of lateral flow immunosensors for IL-6 is innovative, as we could not find patents describing such a biosensor. Scientific prospection reported two lateral flow immunosensors. However, the test for IL-6 cannot be completed without using some kind of equipment for detection, such as the one we propose in future developments.


Protonophore- and pH-insensitive glucose and sucrose accumulation detected by FRET nanosensors in Arabidopsis root tips.
Although soil contains only traces of soluble carbohydrates, plant roots take up glucose and sucrose efficiently when supplied in artificial media. Soluble carbohydrates and other small metabolites found in soil are in part products from exudation from plant roots. The molecular nature of the transporters for uptake and exudation is unknown. Here, fluorescence resonance energy transfer (FRET) glucose and sucrose sensors were used to characterize accumulation and elimination of glucose and sucrose in Arabidopsis roots tips. Using an improved image acquisition set-up, FRET responses to perfusion with carbohydrates were detectable in roots within less than 10 sec and over a wide concentration range. Accumulation was fully reversible within 10-180 sec after glucose or sucrose had been withdrawn; elimination may be caused by metabolism and/or efflux. The rate of elimination was unaffected by pre-incubation with high concentrations of glucose, suggesting that elimination is not due to accumulation in a short-term buffer such as the vacuole. Glucose and sucrose accumulation was insensitive to protonophores, was comparable in media differing in potassium levels, and was similar at pH 5.8, 6.8 and 7.8, suggesting that both influx and efflux may be mediated by proton-independent transport systems. High-resolution expression mapping in root tips showed that only a few proton-dependent transport of the STP (Sugar Transport Protein) and SUT/SUC (Sucrose Transporter/Carrier) families are expressed in the external cell layers of root tips. The root expression maps may help to pinpoint candidate genes for uptake and release of carbohydrates from roots.


Automatic image acquisition, calibration and montage assembly for biological X-ray microscopy.
We describe a system for the automatic acquisition and processing of digital images in a high-resolution X-ray microscope, including the formation of large-field high-resolution image montages. A computer-controlled sample positioning stage provides approximate coordinates for each high-resolution subimage. Individual subimages are corrected to compensate for time-varying, non-uniform illumination and CCD-related artefacts. They are then automatically assembled into a montage. The montage assembly algorithm is designed to use the overlap between each subimage and multiple neighbours to improve the performance of the registration step and the fidelity of the result. This is accomplished by explicit use of recorded stage positions, optimized ordering of subimage insertion, and registration of subimages to the developing montage. Using this procedure registration errors are below the resolution limit of the microscope (43 nm). The image produced is a seamless, large-field montage at full resolution, assembled automatically without human intervention. Beyond this, it is also an accurate X-ray transmission map that allows the quantitative measurement of anatomical and chemical features of the sample. Applying these tools to a biological problem, we have conducted the largest X-ray microscopical study to date.


Allosteric regulation of [3H]vinblastine binding to P-glycoprotein of MCF-7 ADR cells by dexniguldipine.
Plasma membranes were prepared from the P-glycoprotein expressing human breast cancer cell line MCF-7 ADR. [3H]Vinblastine bound to these membranes saturably with a Bmax of 24 pmol/mg of protein and KD of 23 nM. In contrast, membranes from the parent cells MCF-7 WT, which do not express P-glycoprotein, did not bind [3H]vinblastine with high affinity. Cytotoxics known to be transported by P-glycoprotein inhibited the binding of [3H]vinblastine, as did multidrug reversing agents including the 1,4-dihydropyridine, dexniguldipine-HCl (Ki, 15 nM). In dissociation kinetic experiments, dexniguldipine-HCl accelerated the dissociation of [3H]vinblastine from P-glycoprotein, indicating a negative heterotropic allosteric mechanism of action through a drug binding site distinct from that of vinblastine. Other 1,4-dihydropyridines tested also accelerated [3H]vinblastine dissociation from P-glycoprotein, however, multidrug reversing drugs of different chemical classes, including quinidine, verapamil and cyclosporin A did not. These results suggest that P-glycoprotein of MCF-7 ADR cell membranes possesses at least two drug acceptor sites which are allosterically coupled: receptor site-1 which binds vinca alkaloids, and receptor site-2 which binds 1,4-dihydropyridines such as dexniguldipine-HCl, which had the highest affinity of the tested derivatives.


5D Flow MRI: A Fully Self-gated, Free-running Framework for Cardiac and Respiratory Motion-resolved 3D Hemodynamics.
To implement, validate, and apply a self-gated free-running whole-heart five-dimensional (5D) flow MRI framework to evaluate respiration-driven effects on three-dimensional (3D) hemodynamics in a clinical setting. In this prospective study, a free-running five-dimensional (5D) flow sequence was implemented with 3D radial sampling, self-gating, and a compressed-sensing reconstruction. The 5D flow was evaluated in a pulsatile phantom and adult participants with aortic and/or valvular disease who were enrolled between May and August 2019. Conventional twofold-accelerated four-dimensional (4D) flow of the thoracic aorta with navigator gating was performed as a reference comparison. Continuous parameters were evaluated for parameter normality and were compared between conventional 4D flow and 5D flow using a signed-rank or two-tailed paired t test. Differences between respiratory states were evaluated using a repeated-measure analysis of variance or a nonparametric Friedman test. A total of 20 adult participants (mean age, 49 years ± 17 [standard deviation]; 18 men and two women) were included. In vitro 5D flow results showed excellent agreement with conventional 4D flow-derived values (peak and net flow, <7% difference over all quantified planes). Whole-heart 5D flow data were collected in all participants in 7.65 minutes ± 0.35 (acceleration rate = 36.0-76.9) versus 9.88 minutes ± 3.17 for conventional aortic 4D flow. In vivo, 5D flow demonstrated moderate agreement with conventional 4D flow but demonstrated overestimation in net flow and peak velocity (up to 26% and 12%, respectively) in the ascending aorta and underestimation (<12%) in the arch and descending aorta. Respiratory-resolved analyses of caval veins showed significantly increased net and peak flow in the inferior vena cava in end inspiration compared with end expiration, and the opposite trend was shown in the superior vena cava. A free-running 5D flow MRI framework consistently captured cardiac and respiratory motion-resolved 3D hemodynamics in less than 8 minutes. Supplemental material is available for this article. © RSNA, 2020.


Peripheral NT3 signaling is required for ETS protein expression and central patterning of proprioceptive sensory afferents.
To study the role of NT3 in directing axonal projections of proprioceptive dorsal root ganglion (DRG) neurons, NT3(-/-) mice were crossed with mice carrying a targeted deletion of the proapoptotic gene Bax. In Bax(-/-)/NT3(-/-) mice, NT3-dependent neurons survived and expressed the proprioceptive neuronal marker parvalbumin. Initial extension and collateralization of proprioceptive axons into the spinal cord occurred normally, but proprioceptive axons extended only as far as the intermediate spinal cord. This projection defect is similar to the defect in mice lacking the ETS transcription factor ER81. Few if any DRG neurons from Bax(-/-)/NT3(-/-) mice expressed ER81 protein. Expression of a NT3 transgene in muscle restored DRG ER81 expression in NT3(-/-) mice. Finally, addition of NT3 to DRG explant cultures resulted in induction of ER81 protein. Our data indicate that NT3 mediates the formation of proprioceptive afferent-motor neuron connections via regulation of ER81.


Rhombohedral Multilayer Graphene: A Magneto-Raman Scattering Study.
Graphene layers are known to stack in two stable configurations, namely, ABA or ABC stacking, with drastically distinct electronic properties. Unlike the ABA stacking, little has been done to experimentally investigate the electronic properties of ABC graphene multilayers. Here, we report on the first magneto optical study of a large ABC domain in a graphene multilayer flake, with ABC sequences exceeding 17 graphene sheets. ABC-stacked multilayers can be fingerprinted with a characteristic electronic Raman scattering response, which persists even at room temperatures. Tracing the magnetic field evolution of the inter Landau level excitations from this domain gives strong evidence for the existence of a dispersionless electronic band near the Fermi level, characteristic of such stacking. Our findings present a simple yet powerful approach to probe ABC stacking in graphene multilayer flakes, where this highly degenerated band appears as an appealing candidate to host strongly correlated states.


COX-2 inhibition improves immunotherapy and is associated with decreased numbers of myeloid-derived suppressor cells in mesothelioma. Celecoxib influences MDSC function.
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that accumulates in tumour-bearing hosts. These cells are induced by tumour-derived factors (e.g. prostaglandins) and have a critical role in immune suppression. MDSC suppress T and NK cell function via increased expression of arginase I and production of reactive oxygen species (ROS) and nitric oxide (NO). Immune suppression by MDSC was found to be one of the main factors for immunotherapy insufficiency. Here we investigate if the in vivo immunoregulatory function of MDSC can be reversed by inhibiting prostaglandin synthesis by specific COX-2 inhibition focussing on ROS production by MDSC subtypes. In addition, we determined if dietary celecoxib treatment leads to refinement of immunotherapeutic strategies. MDSC numbers and function were analysed during tumour progression in a murine model for mesothelioma. Mice were inoculated with mesothelioma tumour cells and treated with cyclooxygenase-2 (COX-2) inhibitor celecoxib, either as single agent or in combination with dendritic cell-based immunotherapy. We found that large numbers of infiltrating MDSC co-localise with COX-2 expression in those areas where tumour growth takes place. Celecoxib reduced prostaglandin E2 levels in vitro and in vivo. Treatment of tumour-bearing mice with dietary celecoxib prevented the local and systemic expansion of all MDSC subtypes. The function of MDSC was impaired as was noticed by reduced levels of ROS and NO and reversal of T cell tolerance; resulting in refinement of immunotherapy. We conclude that celecoxib is a powerful tool to improve dendritic cell-based immunotherapy and is associated with a reduction in the numbers and suppressive function of MDSC. These data suggest that immunotherapy approaches benefit from simultaneously blocking cyclooxygenase-2 activity.


Impact of demographic and clinical parameters on video capsule transit time.
Small bowel (SB) capsule endoscopy (CE) studies provide data on both gastric and SB transit times (GTT and SBTT, respectively). This study aimed to evaluate the influence of demographic and clinical parameters on the GTT and SBTT. Transit times for two generations of capsules (Pillcam SB2 and SB3) were also compared. Consecutive adult patients undergoing CE were included. GTT, SBTT, and cecum arrival rates were calculated and correlated to demographics and clinical characteristics. A total of 332 CE studies were analyzed. Neither GTT nor SBTT were impacted by age or sex. SBTT was prolonged in newly diagnosed Crohn's disease (CD) patients compared with all other patients (303.1±90.3 vs. 243.6±83.6 min, P=0.02 for SB2, 267.8±63 vs. 228.6±72.3, P=0.01 for SB3, respectively). Moreover, CD patients had higher incomplete study rates compared with patients with all other diagnoses (29.4 vs. 7.3%, respectively, P=0.0116) in the SB2 subgroup. Higher cecum arrival rates were achieved by the SB3 capsule compared with SB2 (97 vs. 91%, P=0.04). Patients with prolonged gastric time or patients with incomplete studies had similar demographic and clinical characteristics as others. Age and sex apparently do not influence intestinal kinetics. Newly diagnosed CD patients have relatively prolonged SBTTs. Demographic and clinical parameters cannot predict prolonged GTT or cecum nonarrival.


Limitations of improved nitrogen management to reduce nitrate leaching and increase use efficiency.
The primary mode of nitrogen (N) loss from tile-drained row-cropped land is generally nitrate-nitrogen (NO3-N) leaching. Although cropping, tillage, and N management practices can be altered to reduce the amount of leaching, there are limits as to how much can be done. Data are given to illustrate the potential reductions for individual practices such as rate, method, and timing of N applications. However, most effects are multiplicative and not additive; thus it is probably not realistic to hope to get overall reductions greater than 25 to 30% with in-field practices alone. If this level of reduction is insufficient to meet water quality goals, additional off-site landscape modifications may be necessary.


Characterization of T-cell subsets infiltrating post-burn hypertrophic scar tissues.
In this study, skin-infiltrating cells were characterized in both the active and remission phases of post-burn hypertrophic scar biopsies. Immunohistochemistry examination of active phase samples showed an abundant presence of Langerhans cells, T cells, macrophages, a low presence of natural killer cells and the lack of B lymphocytes. In active hypertrophic scars T lymphocytes infiltrate deep into the superficial dermis and are also observed in the epidermis: CD3+ cells were present at about 222 +/- 107 per 0.25 mm2. In particular the analysis of lymphocyte subpopulations showed that CD4+ T cells predominate in the dermis as well as in the epidermis of active hypertrophic scars whereas CD8+ cells were less well represented (CD4/CD8 ratio is 2.06). This distribution was also shown in remission phase samples and in normotrophic scar specimens, although the lymphocyte number was significantly lower. Approximately 70 per cent of T lymphocytes present in the tissue involved in active phase hypertrophic scar samples were activated (positive with anti-HLA-DR and IL-2 receptor antibodies) which is significantly higher than remission phase hypertrophic and normotrophic scars, in which positivity was 40 and 38 per cent, respectively. Upon activation, the lesional lymphocytes release several cytokines, locally and transiently, that interact with specific receptors in response to different stimulation. Central to the immune hypothesis of hypertrophic scars is that some of the T-cell lymphokines act on keratinocytes, fibroblasts and other cell types to induce changes characteristic of these scars. The presence and close proximity of activated T lymphocytes and antigen-presenting cells of various phenotypes in both the epidermis and dermis of hypertrophic tissues provides strong circumstantial evidence of a local immune response. However, the manner in which T cells achieve and maintain their activated state in hypertrophic tissues is not yet known, and both antigen-dependent and independent mechanisms may contribute.


Increasing AIP Macrocycle Size Reveals Key Features of agr Activation in Staphylococcus aureus.
The agr locus in the commensal human pathogen, Staphylococcus aureus, is a two-promoter regulon with allelic variability that produces a quorum-sensing circuit involved in regulating virulence within the bacterium. Secretion of unique autoinducing peptides (AIPs) and detection of their concentrations by AgrC, a transmembrane receptor histidine kinase, coordinates local bacterial population density with global changes in gene expression. The finding that staphylococcal virulence can be inhibited through antagonism of this quorum-sensing pathway has fueled tremendous interest in understanding the structure-activity relationships underlying the AIP-AgrC interaction. The defining structural feature of the AIP is a 16-membered, thiolactone-containing macrocycle. Surprisingly, the importance of ring size on agr activation or inhibition has not been explored. In this study, we address this deficiency through the synthesis and functional analysis of AIP analogues featuring enlarged and reduced macrocycles. Notably, this study is the first to interrogate AIP function by using both established cell-based reporter gene assays and newly developed in vitro AgrC-I binding and autophosphorylation activity assays. Based on our data, we present a model for robust agr activation involving a cooperative, three-points-of-contact interaction between the AIP macrocycle and AgrC.


Effects of saturation on the optical scintillometer.
Measurements of the level of turbulence C(2)(n) have been successfully performed with the optical scintillometer. The success of this instrument is based on the observed fact that the variance of aperture averaged scintillation is described by weak scattering theory even for conditions in which strong scintillation is observed for point detectors. However, for sufficiently long propagation paths, the aperture averaged variance is affected by strong scattering. The effects of strong scattering are calculated theoretically and compared to experimental results. The physics of this regime are discussed and the important parameters investigated. The new range of validity of the optical scintillometer is discussed.


Periodontal conditions in Switzerland at the end of the 20th century.
To epidemiologically assess the periodontal conditions of the adult population in Switzerland. During a ten-year period, a total of 1318 subjects were randomly selected on the basis of a process involving the community rosters in seven regions (Cantons) of Switzerland: (Canton of Berne; Canton of Zurich; Western cantons (GE,VD,NE); Eastern cantons (SG,AI,AR,TG,SH); Southern cantons (VS,GR,TI); Central cantons (LU,GL,ZG,UR,SZ,OW,NW) and Northern cantons (JU,FR,SO,AG,BL)) and encompassing all adult age groups (20-89 years). The subjects were examined in dental offices randomly distributed throughout the country. The number of teeth present, the mean Plaque and Gingival as well as Retention Indices were assessed. Furthermore, pocket probing depths and loss of periodontal attachment were determined on all teeth. Ninety-four (7.1%) of the subjects were completely edentulous leaving 1224 dentate individuals with an average of 21.65 teeth for analysis. In the youngest cohort (20-29 years) 27.03 teeth were present. During the fourth to the seventh decade of life, tooth loss appeared to follow a linear pattern leaving 17.63 teeth in the age group of 60-69 years. The oldest age group of the 80-89 years old yielded 11.08 teeth. The mean scores of all clinical parameters increased significantly with increasing age. Mean PII in the youngest age group was PII: 0.72 (SD: 0.38) and reached PII: 1.55 (SD: 0.68) in the oldest. The corresponding mean GI were 1.17 (SD: 0.31) and GI: 1.64 (SD: 0.50), respectively. The mean RI increased from 0.24 (SD: 0.29) to RI: 1.34 (SD: 0.70) in the oldest. Mean probing depth increased slightly from 20 to approximately 49 years. Thereafter, mean PPD remained around 3.0 mm. The loss of periodontal attachment increased dramatically after the age of 50. Periodontitis manifests itself after the age of 50. The pronounced progressive attachment loss in the age cohorts over fifty years explained the marked loss of teeth in some individuals. From a public health point of view, it is indicated to make a major effort for periodontal prevention in the population approaching the second third of life.


Therapeutic options for the management of hot flashes in breast cancer survivors: an evidence-based review.
Women with breast cancer may experience treatment-induced menopausal symptoms or natural menopause. Menopausal symptoms, particularly hot flashes, are reported at a high frequency in this group and tend to be more severe, distressing, and of greater duration than in controls. Because of the contribution of sex hormones to breast cancer, the use of hormonal agents for the control of hot flashes is problematic in these women. Safer nonhormonal alternatives are recommended for this patient group. This was a systematic review of the therapeutic options for the treatment of hot flashes in breast cancer survivors. MEDLINE was searched from 1990 to July 2006 using the disease-specific term breast neoplasms and the subheadings menopause and hot flashes. EMBASE was searched from 1990 to March 2006 using the disease-specific subject headings breast tumor/ breast cancer and menopause and the key word hot flashes. The reference lists of the identified articles and relevant review articles were examined for additional publications. Pertinent articles and abstracts of large randomized controlled trials focusing on the treatment of hot flashes in breast cancer survivors were selected for review. Pilot studies were excluded. A number of nonpharmacologic approaches are available for the treatment of hot flashes in breast cancer survivors, although they appear to be of limited effectiveness. Complementary alternative medicine therapies and vitamin E have been found to have modest effectiveness at best, and data on their long-term safety are not available. Centrally active agents such as the antidepressants venlafaxine and paroxetine and the anti seizure agent gabapentin have shown clinical effectiveness and appear to be reasonably well tolerated in this population. Centrally active agents (eg, venlafaxine, paroxetine, gabapentin) are regarded as the most promising nonhormonal treatments for hot flashes in breast cancer survivors. Nonpharmacologic and complementary alternative medicine therapies have limited effectiveness.


The effects of foundation species on community assembly: a global study on alpine cushion plant communities.
Foundation species can change plant community structure by modulating important ecological processes such as community assembly, yet this topic is poorly understood. In alpine systems, cushion plants commonly act as foundation species by ameliorating local conditions. Here, we analyze diversity patterns of species' assembly within cushions and in adjacent surrounding open substrates (83 sites across five continents) calculating floristic dissimilarity between replicate plots, and using linear models to analyze relationships between microhabitats and species diversity. Floristic dissimilarity did not change across biogeographic regions, but was consistently lower in the cushions than in the open microhabitat. Cushion plants appear to enable recruitment of many relatively stress-intolerant species that otherwise would not establish in these communities, yet the niche space constructed by cushion plants supports a more homogeneous composition of species than the niche space beyond the cushion's influence. As a result, cushion plants support higher α-diversity and a larger species pool, but harbor assemblies with lower β-diversity than open microhabitats. We conclude that habitats with and without dominant foundation species can strongly differ in the processes that drive species recruitment, and thus the relationship between local and regional species diversity.


Unmet need for contraception among HIV-positive women in Lesotho and implications for mother-to-child transmission.
In Lesotho, the risk of mother-to-child-transmission (MTCT) of HIV is substantial; women of childbearing age have a high HIV prevalence rate (26.4%), low knowledge of HIV status and a total fertility rate of 3.5 births per woman. An effective means of preventing MTCT is to reduce unwanted fertility. This paper examines the unmet need for contraception to limit and space births among HIV-positive women in Lesotho aged 15-49 years, using the 2004 Lesotho Demographic and Health Survey. HIV-positive women have their need for contraception unmet in almost one-third of cases, and multivariate analysis reveals this unmet need is most likely amongst the poor and amongst those not approving of family planning. Urgent action is needed to lower the level of unmet need and reduce MTCT. A constructive strategy is to improve access to family planning for all women in Lesotho, irrespective of HIV status, and, more specifically, integrate family planning with MTCT prevention and voluntary counselling and testing services.


In vivo antitumor activity of various forms of delayed-type hypersensitivity in mice.
Relationships among various forms of delayed-type hypersensitivity (DTH) and nonspecific resistance to Lewis lung tumor were studied in syngeneic and semisyngeneic mice. Only the tuberculin type of DTH obviated a virulent inoculum of 10(6) tumor cells. The Jones-Mote type of DTH, even modified by cyclophosphamide pretreatment, produced a significant local inflammatory reaction which was unable to destroy tumor cells. The antitumor effect of the tuberculin type was observed in BCG-or in Corynbacterium parvum-immune mice and also in sheep red blood cell-immunized mice, but only after the modulating effect of BCG. The antitumor activity of the DTH reaction was anatomically restricted and time related, and it required local persistence of specific antigen. A minimal number of bacteria, 1 times 10(6) living or heat-killed BCG organisms, were equally able to eradicate 10(5) tumor cells in BCG-immune mice. Biphasic effects on tumor growth were observed when systemic specific inflammatory reactions were elicited in BCG-immune mice. However, tumor-specific immunity was never observed, inasmuch as BCG-immune mice surviving injection of a mixture of BCG and tumor cells did not resist a second tumor cell challenge.


B7-H3 promotes acute and chronic allograft rejection.
The B7 homolog B7-H3 is important for the regulation of immune responses though its functions in vivo are controversial. We report the first clinical and experimental data concerning expression and function of B7-H3 in alloresponses. Immunohistological and molecular analyses showed B7-H3 expression by cells mediating rejection of human and mouse allografts. To analyze the significance of B7-H3 in rejecting allografts, we generated B7-H3-/- mice and showed that targeting of B7-H3 was synergistic with other forms of immune modulation; e.g. a regimen of rapamycin gave 12-14 days of survival in wild-type controls but led to permanent cardiac and islet allograft survival in B7-H3-/- mice. Cardiac allografts in treated B7-H3-/- mice showed markedly decreased production of key cytokine, chemokine and chemokine receptor mRNA transcripts as compared to wild-type controls. The incidence of chronic rejection in two different cardiac allograft models was also inhibited in B7-H3-/- as compared to wild-type recipients. Lastly, in addition to the expected antigen-presenting cell expression of B7-H3, CD4 and CD8 T cells showed B7-H3 induction upon cell activation, and both dendritic cell- and T cell-expressed B7-H3 each enhanced T cell proliferation in vitro and in vivo. We conclude that B7-H3 promotes T cell-mediated immune responses and the development of acute and chronic allograft rejection.


Comparison of Treatment Outcomes of Surgical Repair in Inguinal Hernia with Classic versus Preperitoneal Methods on Reduction of Postoperative Complications.
Background. This study aims to evaluate and compare the results of inguinal herniorrhaphy with mesh in classic and preperitoneal method. Methods. Our study community includes 150 candidate patients for inguinal herniorrhaphy with mesh. Totally, 150 candidate patients for inguinal herniorrhaphy were randomly divided into two groups: (1) classic group in which the floor of the canal was repaired and the mesh was located on the floor of the canal and (2) preperitoneal group in which the mesh was installed under the canal and then the floor was repaired. Results. The frequency of recurrence was 10 (13.3%) and 2 (2.66%) in the classic and preperitoneal group, respectively. The frequency of postsurgical pain was 21 (28%) in the classic group and 9 (12%) in the preperitoneal group. The postsurgical hematoma was observed in 7 (9.3%) and 9 (12%) in the classic and preperitoneal group, respectively. Also, the frequency of postsurgical seroma was 8 (10.7%) and 1 (1.3%) in the patients treated with the classic and preperitoneal method, respectively. Conclusion. The findings of the present study demonstrated that the preperitoneal method is a more suitable method for inguinal herniorrhaphy than the classic one because of fewer complications, according to the findings of this study.


ConceptVector: Text Visual Analytics via Interactive Lexicon Building Using Word Embedding.
Central to many text analysis methods is the notion of a concept: a set of semantically related keywords characterizing a specific object, phenomenon, or theme. Advances in word embedding allow building a concept from a small set of seed terms. However, naive application of such techniques may result in false positive errors because of the polysemy of natural language. To mitigate this problem, we present a visual analytics system called ConceptVector that guides a user in building such concepts and then using them to analyze documents. Document-analysis case studies with real-world datasets demonstrate the fine-grained analysis provided by ConceptVector. To support the elaborate modeling of concepts, we introduce a bipolar concept model and support for specifying irrelevant words. We validate the interactive lexicon building interface by a user study and expert reviews. Quantitative evaluation shows that the bipolar lexicon generated with our methods is comparable to human-generated ones.


Selective protein depletion impairs bone growth and causes liver fatty infiltration in female rats: prevention by Spirulina alga.
Chronic protein malnutrition leads to child mortality in developing countries. Spirulina alga (Spi), being rich in protein and growing easily, is a good candidate as supplementation. We showed that Spi completely prevents bone growth retardation and liver disturbances observed in young rats fed a low protein diet. This supports Spi as a useful source of vegetable protein to fight against protein malnutrition. Chronic malnutrition is a main factor of child mortality in developing countries. A low protein diet impairs whole-body growth and leads to fatty liver in growing rats. Spi has great potential as a supplementation as it has a 60 % protein content and all essential amino acids. However, its specific impact on bone growth and the related secretion of hepatokines have not yet been studied. To address this question, 6-week-old female rats were fed isocaloric diets containing 10 % casein, 5 % casein, or 5 % casein + 5 % protein from Spi during 9 weeks. Changes in tibia geometry, microarchitecture, BMC, BMD, and biomechanical properties were analyzed. Serum IGF-I, FGF21, follistatin, and activin A were assessed as well as their hepatic gene expressions in addition to those of Sirt1, Ghr, and Igf1r. Hepatic fat content was also assessed. A low protein diet altered bone geometry and reduced proximal tibia BMD and trabecular bone volume. In addition, it increased hepatic fat content and led to hepatic GH resistance by decreasing serum IGF-I and increasing serum FGF21 without altering serum activin A and follistatin. Spi prevented low protein diet-induced bone, hepatic, and hormonal changes, and even led to higher biomechanical properties and lower hepatic fat content in association with specific InhbA and Follistatin expression changes vs. the 10 % casein group. Altogether our results demonstrate the preventive impact of Spi on bone growth delay and hepatic GH resistance in conditions of isocaloric dietary protein deficiency.


A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.
Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.


Unattainable equipoise in randomized controlled trials : staff views of a feasibility study of surgical treatments for segmental tibial fractures.
To explore staff experiences of a multicentre pilot randomized controlled trial (RCT) comparing intramedullary nails and circular frame external fixation for segmental tibial fractures. A purposeful sample of 19 staff (nine surgeons) involved in the study participated in an interview. Interviews explored participants' experience and views of the study and the treatments. The interviews drew on phenomenology, were face-to-face or by telephone, and were analyzed using thematic analysis. The findings identify that for the treatment of segmental tibial fractures equipoise was a theoretical ideal that was most likely unattainable in clinical practice. This was conveyed through three themes: the ambiguity of equipoise, where multiple definitions of equipoise and a belief in community equipoise were evident; an illusion of equipoise, created by strong treatment preferences and variation in collective surgical skills; and treating the whole patient, where the complexity and severity of the injury required a patient-centred approach and doing the best for the individual patient took priority over trial recruitment. Equipoise can be unattainable for rare injuries such as segmental tibial fractures, where there are substantially different surgical treatments requiring specific expertise, high levels of complexity, and a concern for poor outcomes. Surgeons are familiar with community equipoise. However, a shared understanding of factors that limit the feasibility of RCTs may identify instances where community equipoise is unlikely to translate into practice. Cite this article: Bone Jt Open 2021;2(7):486-492.


Acute and Cumulative Effects of Vinyasa Yoga on Affect and Stress among College Students Participating in an Eight-week Yoga Program: A Pilot Study.
College students are vulnerable to a critical period in developmental maturation, facing rigorous academic work and learning how to function independently. Western aerobic exercise (WAE), such as running and bicycling, has been shown to improve mood and relieve stress. However, college students often have low levels of physical activity. Yoga is an ancient physical and mental practice that may affect mood and stress. However, rigorous studies examining the psychological effects of yoga are rare in peerreviewed Western journals. The aim of this research was to establish preliminary evidence for the acute effects of Vinyasa yoga on affect and stress in young-adult college students. Twenty healthy college students age 18 years and older were recruited to participate in this pilot study. Participants attended a Vinyasa yoga class at a local studio twice weekly for 8 weeks. Affect and stress were assessed before and after each yoga session. Measures included the Positive and Negative Affective Schedule (PANAS) and the Cohen Perceived Stress scale. Positive affect scores increased significantly (p < 0.05) for 14 of the 16 yoga sessions (mean increase = 23.2%). Negative affect decreased significantly from pre- to postyoga (p < 0.05) for 15 of the 16 sessions (mean decrease = 22%). Repeated measures ANOVAs examining pre-post composite scores across all 8 weeks showed significant changes in PANAS, but not stress scores. Findings suggest that yoga practice is associated with acute improvements in affect in a young-adult college population. Future research is needed to examine the extent to which different types of yoga address the needs of different college sub-populations (e.g., eating disordered, overweight/obese, sedentary, and smokers).


Factors influencing use of an e-health website in a community sample of older adults.
The use of the internet as a source of health information and link to healthcare services has raised concerns about the ability of consumers, especially vulnerable populations such as older adults, to access these applications. This study examined the influence of training on the ability of adults (aged 45+ years) to use the Medicare.gov website to solve problems related to health management. The influence of computer experience and cognitive abilities on performance was also examined. Seventy-one participants, aged 47-92, were randomized into a Multimedia training, Unimodal training, or Cold Start condition and completed three healthcare management problems. MEASUREMENT AND ANALYSES: Computer/internet experience was measured via questionnaire, and cognitive abilities were assessed using standard neuropsychological tests. Performance metrics included measures of navigation, accuracy and efficiency. Data were analyzed using analysis of variance, χ(2) and regression techniques. The data indicate that there was no difference among the three conditions on measures of accuracy, efficiency, or navigation. However, results of the regression analyses showed that, overall, people who received training performed better on the tasks, as evidenced by greater accuracy and efficiency. Performance was also significantly influenced by prior computer experience and cognitive abilities. Participants with more computer experience and higher cognitive abilities performed better. The findings indicate that training, experience, and abilities are important when using complex health websites. However, training alone is not sufficient. The complexity of web content needs to be considered to ensure successful use of these websites by those with lower abilities.


Polarization-dependent electric potential distribution across nanoscale ferroelectric Hf0.5Zr0.5O2 in functional memory capacitors.
The emergence of ferroelectricity in nanometer-thick films of doped hafnium oxide (HfO2) makes this material a promising candidate for use in Si-compatible non-volatile memory devices. The switchable polarization of ferroelectric HfO2 controls functional properties of these devices through the electric potential distribution across the capacitor. The experimental characterization of the local electric potential at the nanoscale has not so far been realized in practice. Here, we develop a new methodology which allows us, for the first time, to experimentally quantify the polarization-dependent potential profile across few-nanometer-thick ferroelectric Hf0.5Zr0.5O2 thin films. Using a standing-wave excitation mode in synchrotron based hard X-ray photoemission spectroscopy, we depth-selectively probe TiN/Hf0.5Zr0.5O2/W prototype memory capacitors and determine the local electrostatic potential by analyzing the core-level line shifts. We find that the electric potential profile across the Hf0.5Zr0.5O2 layer is non-linear and changes with in situ polarization switching. Combined with our scanning transmission electron microscopy data and theoretical modeling, we interpret the observed non-linear potential behavior in terms of defects in Hf0.5Zr0.5O2, at both interfaces, and their charge state modulated by the ferroelectric polarization. Our results provide an important insight into the intrinsic electronic properties of HfO2 based ferroelectric capacitors and are essential for engineering memory devices.


Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention.
Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors.


A genome-wide map of mitochondrial DNA recombination in yeast.
In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance.


Understanding dengue virus evolution to support epidemic surveillance and counter-measure development.
Dengue virus (DENV) causes a profound burden of morbidity and mortality, and its global burden is rising due to the co-circulation of four divergent DENV serotypes in the ecological context of globalization, travel, climate change, urbanization, and expansion of the geographic range of the Ae.aegypti and Ae.albopictus vectors. Understanding DENV evolution offers valuable opportunities to enhance surveillance and response to DENV epidemics via advances in RNA virus sequencing, bioinformatics, phylogenetic and other computational biology methods. Here we provide a scoping overview of the evolution and molecular epidemiology of DENV and the range of ways that evolutionary analyses can be applied as a public health tool against this arboviral pathogen.


The influence of temperature and salinity on energy partitioning in the marine nematode Diplolaimelloides bruciei.
Measurements of population growth, generation time, fecundity and respiration in laboratory culture have been made, in relation to temperature and salinity, for the nematode Diplolaimelloides bruciei Hopper, a species normally associated with decayed material of the marsh grass Spartina. The intrinsic rate of increase (r) is high: it is related to temperature between 5° and 25°C by a sigmoid function which is steepest between 10° and 15°C, and is maximum at 26‰ salinity. Generation time is related to temperature by a power function and is shortest at 26‰ salinity. The effect of temperature on generation time is consistent with other data for marine nematodes, and the steep slope of r against temperature is largely due to the marked effect of temperature on fecundity. A sex ratio of 2:1 in favour of males is maintained regardless of culture conditions or population density. Respiration increases exponentially with temperature between 5° and 25°C, with a very high Q10 (3.94), but is not affected by salinity. At 30°C respiration is no higher than at 25°C.A high and relatively stable production efficiency (P/A) is maintained between 10 and 30°C with a maximum of 87% at 15°C; there is a stable reproductive effort (Pr/A) of about 10%. At 5°C both these ratios are zero. Data for the harpacticoid copepod Tachidius discipes, derived from the literature, show that this too has a high and stable production efficiency, which may be a characteristic of meiofaunal species in general, but in this species efficiency is relatively high at 5°C. Many features of the energy balance in D. bruciei can be related to an opportunistic mode of life.


Posttraumatic seizures - prevention or not.
Early posttraumatic epilepsies (EPTE) are epileptic attacks that appear in first seven days after brain injury, with incidence of 3-5%. Predictors for development of EPTE are: impressive skull fracture with rupture of dura, intracranial haemorrhage, neurogical deficit (brain contusion), and posttraumatic amnesia longer than 24 hours. It is more common in children than in adolescents and adults. It carries four times increased risk for development of late posttraumatic epilepsy. Aspects of pharmacological prophylaxis was often considered, but scientifically neglected, without clear standings regarding controversial data in literature. Patients with severe head injury, hospitalised at Neurosurgical Hospital and Pediatric Hospital, Clinical Centre University of Sarajevo, in period from 6th of April 1992 till 1st of July 1994, were included in study. Prophylaxis of EPTE was carried out with phenobarbital (2-3 mg/kg) or phenytoin (3 mg/kg) parenterally. Decision was made upon clinical findings. CT scan was done in 13.5% patients, and in 31.9% patients serum concentrations of antiepileptic drugs were monitored. 310 patients aged 0-18 years (105 patients 0-10 years, and 205 patients 11-18 years) were investigated. Predictors of EPTE presented were posttraumatic amnesia longer than 24 hours in 90.6%, neurogical deficit in 86.45%, impressive skull fracture with rupture of dura in 81.3% and intracranial haemorrhage in 40.6%. Only two boys developed EPTE in first 24 hours after injury. This study has showed that use of antiepileptic drugs can decrease incidence of EPTE. However, problem remains, management of injured patients is still highly individualised, based on different experiences of doctors that treat patient, and without clear guidelines.


International consensus conference on stool banking for faecal microbiota transplantation in clinical practice.
Although faecal microbiota transplantation (FMT) has a well-established role in the treatment of recurrent Clostridioides difficile infection (CDI), its widespread dissemination is limited by several obstacles, including lack of dedicated centres, difficulties with donor recruitment and complexities related to regulation and safety monitoring. Given the considerable burden of CDI on global healthcare systems, FMT should be widely available to most centres.Stool banks may guarantee reliable, timely and equitable access to FMT for patients and a traceable workflow that ensures safety and quality of procedures. In this consensus project, FMT experts from Europe, North America and Australia gathered and released statements on the following issues related to the stool banking: general principles, objectives and organisation of the stool bank; selection and screening of donors; collection, preparation and storage of faeces; services and clients; registries, monitoring of outcomes and ethical issues; and the evolving role of FMT in clinical practice,Consensus on each statement was achieved through a Delphi process and then in a plenary face-to-face meeting. For each key issue, the best available evidence was assessed, with the aim of providing guidance for the development of stool banks in order to promote accessibility to FMT in clinical practice.


LADA type diabetes, celiac diasease, cerebellar ataxia and stiff person syndrome. A rare association of autoimmune disorders.
Celiac disease--in its typical form--is a chronic immune-mediated enteropathy with typical clinical symptoms that develops against gliadin content of cereal grains, and is often associated with other autoimmune diseases. In cases of atypical manifestation classic symptoms may be absent or mild, and extra-intestinal symptoms or associated syndromes dominate clinical picture. The authors present a longitudinal follow-up of such a case. A 63-years old woman was diagnosed with epilepsy at the age of 19, and with progressive limb ataxia at the age of 36, which was initially thought to be caused by cerebellar atrophy, later probably by stiff person syndrome. At the age 59, her diabetes mellitus manifested with type 2 diabetic phenotype, but based on GAD positivity later was reclassified as type 1 diabetes. Only the last check-up discovered the celiac disease, retrospectively explaining the entire disease course and neurological symptoms. By presenting this case, the authors would like to draw attention to the fact that one should think of the possibility of celiac disease when cerebellar ataxia, progressive neurological symptoms and diabetes are present at the same time. An early diagnosis may help to delay the progression of disease and help better treatment.


The gene tree delusion.
Higher-level relationships among placental mammals are mostly resolved, but several polytomies remain contentious. Song et al. (2012) claimed to have resolved three of these using shortcut coalescence methods (MP-EST, STAR) and further concluded that these methods, which assume no within-locus recombination, are required to unravel deep-level phylogenetic problems that have stymied concatenation. Here, we reanalyze Song et al.'s (2012) data and leverage these re-analyses to explore key issues in systematics including the recombination ratchet, gene tree stoichiometry, the proportion of gene tree incongruence that results from deep coalescence versus other factors, and simulations that compare the performance of coalescence and concatenation methods in species tree estimation. Song et al. (2012) reported an average locus length of 3.1 kb for the 447 protein-coding genes in their phylogenomic dataset, but the true mean length of these loci (start codon to stop codon) is 139.6 kb. Empirical estimates of recombination breakpoints in primates, coupled with consideration of the recombination ratchet, suggest that individual coalescence genes (c-genes) approach ∼12 bp or less for Song et al.'s (2012) dataset, three to four orders of magnitude shorter than the c-genes reported by these authors. This result has general implications for the application of coalescence methods in species tree estimation. We contend that it is illogical to apply coalescence methods to complete protein-coding sequences. Such analyses amalgamate c-genes with different evolutionary histories (i.e., exons separated by >100,000 bp), distort true gene tree stoichiometry that is required for accurate species tree inference, and contradict the central rationale for applying coalescence methods to difficult phylogenetic problems. In addition, Song et al.'s (2012) dataset of 447 genes includes 21 loci with switched taxonomic names, eight duplicated loci, 26 loci with non-homologous sequences that are grossly misaligned, and numerous loci with >50% missing data for taxa that are misplaced in their gene trees. These problems were compounded by inadequate tree searches with nearest neighbor interchange branch swapping and inadvertent application of substitution models that did not account for among-site rate heterogeneity. Sixty-six gene trees imply unrealistic deep coalescences that exceed 100 million years (MY). Gene trees that were obtained with better justified models and search parameters show large increases in both likelihood scores and congruence. Coalescence analyses based on a curated set of 413 improved gene trees and a superior coalescence method (ASTRAL) support a Scandentia (treeshrews)+Glires (rabbits, rodents) clade, contradicting one of the three primary systematic conclusions of Song et al. (2012). Robust support for a Perissodactyla+Carnivora clade within Laurasiatheria is also lost, contradicting a second major conclusion of this study. Song et al.'s (2012) MP-EST species tree provided the basis for circular simulations that led these authors to conclude that the multispecies coalescent accounts for 77% of the gene tree conflicts in their dataset, but many internal branches of their MP-EST tree are stunted by an order of magnitude or more due to wholesale gene tree reconstruction errors. An independent assessment of branch lengths suggests the multispecies coalescent accounts for ⩽ 15% of the conflicts among Song et al.'s (2012) 447 gene trees. Unfortunately, Song et al.'s (2012) flawed phylogenomic dataset has been used as a model for additional simulation work that suggests the superiority of shortcut coalescence methods relative to concatenation. Investigator error was passed on to the subsequent simulation studies, which also incorporated further logical errors that should be avoided in future simulation studies. Illegitimate branch length switches in the simulation routines unfairly protected coalescence methods from their Achilles' heel, high gene tree reconstruction error at short internodes. These simulations therefore provide no evidence that shortcut coalescence methods out-compete concatenation at deep timescales. In summary, the long c-genes that are required for accurate reconstruction of species trees using shortcut coalescence methods do not exist and are a delusion. Coalescence approaches based on SNPs that are widely spaced in the genome avoid problems with the recombination ratchet and merit further pursuit in both empirical systematic research and simulations.


The influence of social factors on gender health.
Male births exceed female births by 5-6% (for a sex ratio at birth of 1.05-1.06) while a women's life expectancy, on a global scale, is about 6 years longer. Thus within various age groups the male:female ratio changes over time. Until age 50 years men outnumber women; thereafter their numbers show a sharp decline. Consequently at age 80 years, there are many more women than men. An estimated 25% of this male excess mortality is due to biological causes, the rest being explained by behavioural, cultural and environmental factors. For both women and men, the main health risks related to lifestyle are smoking, alcohol, unhealthy diet and physical inactivity. In the year 2010, overweight (BMI: 25-29 kg/m(2)) and obesity (BMI: >30 kg/m(2)) were responsible for over 3 million deaths, with similar relative risks in men and women for overweight and obesity. Smoking and alcohol are the major causes of the global gender gap in mortality. For women in some parts of the world however pregnancy is also hazardous. On a global scale, in 2013 about 300 000 deaths were related to pregnancy, with sub-Saharan Africa registering the highest maternal mortality: over 500 maternal deaths per 100 000 births. Additional woman's health risks arise from gender discrimination, including sex-selective abortion, violence against women and early child marriage. Providers should be aware of the effect that these risks can have on both reproductive and general health.


Distribution of ribonucleic acid coliphages in animals.
To determine the distribution pattern of ribonucleic acid (RNA) coliphages (classified by serological groups I through IV) in animal sources, we isolated RNA phages from (i) feces samples from domestic animals (cows, pigs, horses, and fowls), some other animals in a zoological garden, and humans, (ii) the gastrointestinal contents of cows and pigs, and (iii) sewage samples from treatment plants in slaughter houses. These samples were then analyzed serologically. The concentration of RNA phages in the first and second kinds of material was fairly low (10 to 10(3) plaque-forming units per original phage sample), whereas that in the third kind of material was fairly high (10(3) to 10(5) plaque-forming units per original phage sample). Concerning the group types of the RNA phages in the first and second kinds of material, human feces contained RNA phages of groups II and III almost equally, the gastrointestinal contents of pigs included those of groups I and II equally, and the feces or gastrointestinal contents of other mammals other than humans and pigs had those of group I exclusively. In the third type of material we found mostly group I phages with a minor fraction of group II phages. Thus, the prominent features of the distribution pattern of RNA phages are the predominance of groups III and II in humans and the predominance of group I in animals.


Multiple lineages of tumors express a common tumor antigen, P1A, but they are not cross-protected.
Whether tumors of different lineages share common Ags is a critical issue for understanding anti-tumor immune responses and for designing Ag-specific tumor immunotherapy. Because of lack of cross-protection among individually derived tumors, it has been proposed that tumor Ags are specific for individual tumors. Here we show that lack of cross-protection is not due to lack of a shared tumor Ag. Thus, a plasmocytoma J558 transfected with the costimulatory molecule B7 activates a cross-reactive CTL response in vivo. The major Ag recognized by the cross-reactive CTL is P1A, which is expressed in mastocytoma P815, plasmocytoma J558, and fibrosarcoma Meth A. Surprisingly, no significant cross-protection can be detected among P1A-expressing tumors after immunization with either P1A-expressing or B7-transfected P815 cells. Our results demonstrate that multiple lineages of tumors are not cross-protected even though they share a tumor Ag that can be recognized by CTL. These results have important implications for tumor immunotherapy.


Effect of Maternal Body Mass Index on Postpartum Hemorrhage.
It is unclear whether obesity is a risk factor for postpartum hemorrhage. The authors hypothesized that obese women are at greater risk of hemorrhage than women with a normal body mass index. The authors conducted a cohort study of women who underwent delivery hospitalization in California between 2008 and 2012. Using multilevel regression, the authors examined the relationships between body mass index with hemorrhage (primary outcome), atonic hemorrhage, and severe hemorrhage (secondary outcomes). Stratified analyses were performed according to delivery mode. The absolute event rate for hemorrhage was 60,604/2,176,673 (2.8%). In this cohort, 4% of women were underweight, 49.1% of women were normal body mass index, 25.9% of women were overweight, and 12.7%, 5.2%, and 3.1% of women were in obesity class I, II, and III, respectively. Compared to normal body mass index women, the odds of hemorrhage and atonic hemorrhage were modestly increased for overweight women (hemorrhage: adjusted odds ratio [aOR], 1.06; 99% CI, 1.04 to 1.08; atonic hemorrhage: aOR, 1.07; 99% CI, 1.05 to 1.09) and obesity class I (hemorrhage: aOR, 1.08; 99% CI, 1.05 to 1.11; atonic hemorrhage; aOR, 1.11; 99% CI, 1.08 to 1.15). After vaginal delivery, overweight and obese women had up to 19% increased odds of hemorrhage or atonic hemorrhage; whereas, after cesarean delivery, women in any obesity class had up to 14% decreased odds of severe hemorrhage. The authors' findings suggest that, at most, maternal obesity has a modest effect on hemorrhage risk. The direction of the association between hemorrhage and body mass index may differ by delivery mode.


Aperture effect for LINAC-based SRS in small target treatment†.
Stereotactic Radiosurgery (SRS) can utilize different techniques. For small target SRS, the selections of the apertures of cone or MLC field influence the treatment significantly. In this study, while the aperture of the MLC and cone field vary, the dosimetric characteristics of the two techniques are compared for SRS of small targets which are in the favor of magnifying the dosimetric effect. An anthropomorphic phantom was used to simulate the patient with an arbitrary drawn cranial target, whose volume is equivalent to a 4 millimeter sphere. Single 360 ARC plans were generated with Eclipse external beam and cone based treatment planning system from Varian Medical System. For MLC based plan, the apertures fit to the structure with circle margins varying with 2mm, 3mm, 4mm, 5mm and 6mm. These setups were compared with the cones being selected to be 5mm, 10mm, 12mm, 14mm and 16mm in diameter. For the MLC based plan, the leaf-edge-contour meet points were selected to be in the middle. Jaw positions were used both recommend and optimize options, which are provided by the treatment planning system. Mean dose was treated as the prescription dose. Comparison of the two planning techniques was carried out using 12Gy Volume (V12Gy) from dose-volume histogram (DVH), maximum dose to the prescription dose ratio (MDPD), ratio PITV (PIV/TV), radiation conformity index (RCI), which is defined to be TVPIV/PIV, and an integrated conformity index (TVPIV2/ (TVxPIV)), where PIV is the prescription isodose surface volume, TV is target volume, and TVPIV is the intersection of TV and the PIV. For the apertures used for this study, while MLC and cone based plans were compared by prescribed at 20Gy, the V12Gy ranges were from 1.0cc to 5.3cc with average at 3.3cc, and 0.1cc to 3.2cc with average value at 1.5cc. The variations of MDPD were 9% with average value at 1.04, and 12% with average at 1.03. The varying ranges of PITV were 35% with average value at 0.80, and 17% with average value at 0.54. Integrated conformity index (ICI) variations were up to 45% with average value at 0.34, and at the level of the 7% with average value at 0.42. For small target SRS, there are larger variations in V12Gy, PITV, and ICI in MLC based plan than those of cone based plan, while the difference is not significant for MDPD in both settings at the selected aperture sizes. These information could help in decision making through sensitivity study while sufficient clinical outcome information were available. The multiple ARC treatment plan and the clinical reality of algorithm for small aperture also need further investigation.


Histopathology associated with malposition of the human temporomandibular joint disc.
Anterior displacement of the temporomandibular joint disc is associated with a general remodeling of the joint's soft tissues. The anterior part of the posterior attachment becomes fibrotic. The external shape and the internal architecture of the disc change in characteristic ways. The disc appears to overlie more of the lateral pterygoid muscle than is normal. The capsule connecting the anterior band of the disc to the condyle appears to be elongated.


Monofilament Cerclage Wiring Fixation with Locking Plates for Distal Femoral Fracture: Is it Appropriate?
We aimed to determine the efficacy of cerclage wiring by comparing the clinical and radiological results between internal fixation with locking plates after distal femoral fracture reduction with or without cerclage wiring. One hundred and one patients who received open reduction internal fixation for distal femoral fractures of oblique, spiral, and spiral wedge type between 2007 and 2014 were reviewed retrospectively. Only locking plate fixation was performed in 46 patients, and locking plate fixation with additional cerclage wiring was performed in 55 patients (Group CW). Demographic, clinical, and radiologic factors were evaluated in both the groups. Age, gender, bone mineral density, bone graft, and the presence of concomitant fractures were measured as demographic factors. The range of motion of knee joint, Lysholm knee score, visual analog scale score, procedure time, and C-arm time were measured as clinical factors preoperatively and at the final followup. We also evaluated the duration of bone union and knee joint alignment radiologically. There were no demographic differences between the two groups. Furthermore, there were no statistically significant differences between the two groups in terms of clinical and radiological parameters. However, the procedure time used was significantly longer in Group LP than in Group CW (108.4 vs. 95.2 min; P = 0.027). The C-arm time was longer in Group LP (2.8 vs. 1.2 s; P = 0.017). Open reduction and locking plate fixation with additional cerclage wiring is a useful method for the reduction of complicated distal femoral fractures, without increased complications such as nonunion. Level III, retrospective cohort design, treatment study.


Inheritance and biogenesis of organelles in the secretory pathway.
In eukaryotic cells, cellular functions are compartmentalized into membrane-bound organelles. This has many advantages, as shown by the success of the eukaryotic lineage, but creates many problems for cells, such as the need to build and partition these organelles during cell growth and division. Diverse mechanisms for biogenesis of the endoplasmic reticulum and Golgi apparatus have evolved, ranging from de novo synthesis to the copying of a template organelle. The different mechanisms by which organelles are inherited in yeasts, protozoa and metazoans probably reflect the differences in the structure and copy number of these organelles.


Early hominin landscape use in the Lower Omo Valley, Ethiopia: Insights from the taphonomical analysis of Oldowan occurrences in the Shungura Formation (Member F).
The Oldowan archeological record of the Shungura Formation, Member F (Lower Omo valley, Ethiopia) comprises more than one hundred occurrences distributed within archeological complexes, where multiple small spots were found in association with one or two larger occurrences. Such spatial patterning could reflect hominin spatial behavior, repeated occupations within a single sedimentary unit, or taphonomic and/or collection biases. Here we test these hypotheses by way of a geoarcheological and taphonomical analysis using four criteria to assess the preservation of the lithic assemblages: (1) size composition, (2) artifact abrasion, (3) bone abrasion, and (4) orientations of lithic artifacts and bones (i.e., fabrics). We propose a new model of taphonomically induced spatial patterning where the multiple, small, well circumscribed occurrences result primarily from post-depositional processes and therefore do not reflect any underlying behavioral patterns. The large number of archeological occurrences documented in Member F, therefore, corresponds to a limited number of primary occupations (<10). The archeological occupation is mainly restricted to the lower part of Member F and may reflect a single or a small number of occupation episodes, which were located on previous levees of the paleo-Omo River, in nearby floodplain areas, or on the riverbank. This strongly suggests that most of the knapping activities originally took place close to the river. This preference of the Omo toolmakers for riverine environments could explain the scarcity of archeological material in the upper part of Member F that comprises primarily distal floodplain sedimentary facies.


Simulations of allosteric motions in the zinc sensor CzrA.
The zinc sensing transcriptional repressor Staphylococcus aureus CzrA represents an excellent model system to understand how metal sensor proteins maintain cellular metal homeostasis. Zn(II) binding induces a quaternary structural switch from a "closed" conformation to a more "open" conformation, reducing the DNA binding affinity by 4 orders of magnitude. In this study, we use classical molecular dynamics and quantum mechanical/molecular mechanical molecular dynamics simulations to investigate the molecular basis for the large conformational motions and allosteric coupling free energy (~6 kcal/mol) associated with Zn(II) binding. Our simulations successfully capture the closed to open allosteric switching in DNA bound CzrA on Zn(II) binding. They reveal that zinc binding quenches global conformational sampling by CzrA, whereas DNA binding enhances the mobility of residues in the allosteric metal binding sites. These findings are in close agreement with experiments. We also identify networks of residues involved in correlated and anticorrelated motions that connect the metal binding and DNA binding sites. Our analysis of the essential dynamics shows metal ion binding to be the primary driving force for the quaternary structural change in CzrA. We also show that Zn(II) binding limits the conformational space sampled by CzrA and causes the electrostatic surface potential at the DNA binding interface to become less favorable toward DNA binding. Finally, our simulations provide strong support for a proposed hydrogen-bonding pathway that physically connects the metal binding residue, His97, to the DNA binding interface through the αR helix that is present only in the Zn(II)-bound state. Overall, our simulations provide molecular-level insights into the mechanism of allosteric regulation by CzrA and demonstrate the importance of protein motion in its biological activity.


Corpus callosum length by gestational age as evaluated by fetal MR imaging.
Although suspected corpus callosum abnormality is a common indication for fetal MR imaging, biometric data specific to MR imaging are sparse. We sought to characterize growth in corpus callosum length by EGA with fetal MR imaging. Corpus callosum segments were assessed and overall corpus callosum length was measured and plotted against the EGA for 68 anatomically normal fetal brains ranging in EGA from 18.5 to 37.7 weeks, and linear and polynomial regression models were calculated. The body of the corpus callosum was identified in all fetuses, followed in frequency by the splenium (91.2%), genu (85.3%), and rostrum (32.4%). Measurements of corpus callosum length by MR imaging were in agreement with values established by sonography. A second-degree polynomial function was the best fit for callosal length by EGA. Understanding this normal growth pattern may enhance detection of subtle growth abnormalities.


Impact of pneumococcal conjugate vaccination of infants on pneumonia and influenza hospitalization and mortality in all age groups in the United States.
A seven-valent pneumococcal conjugate vaccine (PCV7) introduced in the United States in 2000 has been shown to reduce invasive pneumococcal disease (IPD) in both vaccinated children and adults through induction of herd immunity. We assessed the impact of infant immunization on pneumococcal pneumonia hospitalizations and mortality in all age groups using Health Care Utilization Project State Inpatient Databases (SID) for 1996 to 2006 from 10 states; SID contain 100% samples of ICD9-coded hospitalization data for the selected states. Compared to a 1996-1997 through 1998-1999 baseline, by the 2005-2006 season, both IPD and pneumococcal pneumonia hospitalizations and deaths had decreased substantially in all age groups, including a 47% (95% confidence interval [CI], 38 to 54%) reduction in nonbacteremic pneumococcal pneumonia (ICD9 code 481 with no codes indicating IPD) in infants <2 years old and a 54% reduction (CI, 53 to 56%) in adults ≥65 years of age. A model developed to calculate the total burden of pneumococcal pneumonia prevented by infant PCV7 vaccination in the United States from 2000 to 2006 estimated a reduction of 788,838 (CI, 695,406 to 875,476) hospitalizations for pneumococcal pneumonia. Ninety percent of the reduction in model-attributed pneumococcal pneumonia hospitalizations occurred through herd immunity among adults 18 years old and older; similar proportions were found in pneumococcal disease mortality prevented by the vaccine. In the first seasons after PCV introduction, when there were substantial state differences in coverage among <5-year-olds, states with greater coverage had significantly fewer influenza-associated pneumonia hospitalizations among children, suggesting that PCV7 use also reduces influenza-attributable pneumonia hospitalizations.


HIV infection disrupts the sympatric host-pathogen relationship in human tuberculosis.
The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV-infected and HIV-negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV-infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host-pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21-infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5-19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5-20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV-infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.


Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.
Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high-pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC-MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in the total number of phosphopeptides detected when analyzing the same number of fractions by identical LC-MS/MS gradients. From 14 HpH fractions, we routinely identified over 30,000 unique phosphopeptide variants, which is more than twice the number of that obtained from SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides, with minor benefits for doubly phosphorylated peptides over that with SCX. Further optimizations in the pooling and concatenation strategy increased the total number of multiphosphorylated peptides detected after HpH fractionation. In conclusion, we provide a basic framework and resource for performing in-depth phosphoproteome studies utilizing off-line basic reversed-phased fractionation. Raw data is available at ProteomeXchange (PXD001404).