The effects of chronic infection with a superantigen-producing virus.
C3H/HeJ mice transmit a mouse mammary tumor virus from mother to pup in milk. The retrovirus infects mice shortly after birth and, when expressed in recipient mice, produces a V beta 14-specific superantigen. The consequences of such expression on V beta 14-bearing T cells are examined in this paper. Most cells bearing V beta 14 and either CD4 or CD8 are eliminated in the thymus. Some V beta 14-bearing cells escape to the periphery, however. Those bearing CD8 are unaffected by expression of the viral superantigen. The percentage of peripheral CD4+ T cells bearing V beta 14 drops with time after birth. In large part this seems to be due to the fact that many of these cells become anergic because of exposure to the viral superantigen. Unlike normal T cells, these anergic cells cannot undergo peripheral postthymic expansion. Consequently, they drop in percentage even during a time when their total numbers are constant.


Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.
The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.


Mechanical and energetic effects of cromakalim on guinea pig left ventricular papillary muscle.
The mechanical and energetic effects of the adenosine triphosphate-sensitive potassium channel opener cromakalim at 50 microM and 100 microM were determined on guinea pig left ventricular papillary muscles and compared with those of low calcium concentration (n = 9 in each group). Both concentrations of cromakalim induced a negative inotropic effect (decrease in maximum shortening velocity at preload only and maximum extent of muscle shortening at preload only, P < .01 and P < .001 at 50 and 100 microM, respectively; maximum isometric active force normalized per cross-sectional area during the isometric twitch, P < .001; positive peak of the isometric force derivative normalized per cross-sectional area, P < .001) and a negative lusitropic effect (decrease in maximum lengthening velocity at preload only and negative peak of the isometric force derivative normalized per cross-sectional area, P < .01 and P < .001 at 50 and 100 microM, respectively). At 100 microM, the decrease in relaxation parameters was more marked than that in contraction parameters under isotonic conditions, as shown by the decrease in the slope of the relationship between peak lengthening velocity and maximum extent of muscle shortening (P < .01); this result reflects an intrinsic negative relaxant effect. No relaxant effect was found under isometric conditions at either concentration. The mechanical effects of low calcium were similar to those of cromakalim 100 microM, which suggests that the drug acted mainly by reducing the cellular calcium entry. Despite the negative mechanical effect of cromakalim, mechanical efficiency was preserved. This could partly explain the cardioprotective effect of cromakalim during ischemia.


Multiscale coupling of mesoscopic- and atomistic-level lipid bilayer simulations.
A multiscale method is presented to bridge between the atomistic and mesoscopic membrane systems. The atomistic model in this case is the united atom dimyristoylphosphatidylcholine membrane system, although the method is completely general. Atomistic molecular dynamics provides the expansion modulus which is used to parametrize a mesoscopic elastic membrane model. The resulting elastic membrane model, including explicit mesoscopic solvent, shows appropriate static and dynamic undulation behaviors. Large membranes of approximately 100 nm in length can then be easily simulated using the mesoscopic membrane system. The critical feedback from the mesoscopic system back down to the atomistic-scale system is accomplished by bridging the stress (or surface tension) of a small region in the mesoscopic membrane to the corresponding atomistic membrane system. Because of long length-scale modes of membranes such as undulation and buckling, the local tension responds differently from the frame tension, when subjected to external perturbations. The effect of these membrane modes is shown for the stress response of a local membrane region and therefore the atomistic membrane system. In addition, certain equilibrium static and dynamic properties of stand-alone and multiscale coupled systems are presented for several different membrane sizes. Although static properties such as two-dimensional pair-correlation function and order parameters show no noticeable discrepancy for the different systems, lipid self-diffusion and the rotational relaxation of lipid dipoles have a strong dependence on the membrane size (or long-wavelength membrane motions), which is properly modeled by the present multiscale method.


Quality assessment of observational studies in a drug-safety systematic review, comparison of two tools: the Newcastle-Ottawa Scale and the RTI item bank.
The study objective was to compare the Newcastle-Ottawa Scale (NOS) and the RTI item bank (RTI-IB) and estimate interrater agreement using the RTI-IB within a systematic review on the cardiovascular safety of glucose-lowering drugs. We tailored both tools and added four questions to the RTI-IB. Two reviewers assessed the quality of the 44 included studies with both tools, (independently for the RTI-IB) and agreed on which responses conveyed low, unclear, or high risk of bias. For each question in the RTI-IB (n=31), the observed interrater agreement was calculated as the percentage of studies given the same bias assessment by both reviewers; chance-adjusted interrater agreement was estimated with the first-order agreement coefficient (AC1) statistic. The NOS required less tailoring and was easier to use than the RTI-IB, but the RTI-IB produced a more thorough assessment. The RTI-IB includes most of the domains measured in the NOS. Median observed interrater agreement for the RTI-IB was 75% (25th percentile [p25] =61%; p75 =89%); median AC1 statistic was 0.64 (p25 =0.51; p75 =0.86). The RTI-IB facilitates a more complete quality assessment than the NOS but is more burdensome. The observed agreement and AC1 statistic in this study were higher than those reported by the RTI-IB's developers.


Comprehensive somatic genome alterations of urachal carcinoma.
Urachal cancer is a rare cancer that develops in the urachus. Because of its rarity, standard treatment therapies for urachal cancer are not established, and chemotherapeutic regimens for bladder cancer have been unsuccessful for patients with urachal cancer. Hence, we aim to understand a systematic molecular characterisation of urachal cancer. We identified somatic single-nucleotide variations (SNVs)/indels and somatic copy number aberrations (SCNAs) in the 17 patients by using whole-exome sequencing (WES) and OncoScan platform (Affymetrix) as follows: tumour-normal paired sequencing (WES, n=10), tumour-only sequencing (WES, n=1; targeted deep sequencing, n=16), and OncoScan (n=17). Our analyses identified 27 genes with somatic SNVs and indels, as well as six genes (APC, COL5A1, KIF26B, LRP1B, SMAD4 and TP53) that were recurrent in at least two patients. By analysing the SCNAs, we found that the extent of chromosomal amplification was highly associated with the patient's cancer stage. Interestingly, 35% (6/17) of the patients had focal DNA amplifications in fibroblast growth factor receptor family genes. The integration of somatic SNVs, indels and SCNAs revealed significant alterations in the mitogen-activated protein kinase signalling pathways. Our genome-wide analysis of urachal cancer suggests that molecular characteristics may be important for the treatment of urachal cancer.


Dietary Intake, Body Composition, and Clinical Parameters: Associations Between the Level and Type of Physical Activity in Hemodialysis Patients.
Physical inactivity and muscle wasting potentiate each other and are highly prevalent among hemodialysis (HD) patients. The authors evaluated the association between physical activity (PA), clinical, nutritional, and body composition parameters in HD patients. Multicenter cross-sectional study with 581 HD patients. Clinical, body composition, dietary intake, and PA data were recorded. For the analysis, patients were divided into active (follow World Health Organization recommendations) and inactive groups. A total of 20% of the patients followed World Health Organization recommendations on PA. Differences between physically active and physically inactive patients were observed in age, biochemical parameters and total body water, intracellular water, lean tissue index (LTI), body cell mass, energy, and protein intake. PA was a predictor of higher LTI, body cell mass, and energy intake independently of age, gender, presence of diabetes, dialysis adequacy, and dialysis vintage. Controlling for the effect of age, walking and vigorous PA were positively correlated with energy and protein intake. Vigorous PA was also positively correlated with LTI. The PA is a predictor of higher LTI, body cell mass, and energy intake. Vigorous PA is associated with an improved body composition and dietary pattern, whereas walking seems to be also associated with a favorable nutritional status.


Interferon-gamma receptor signaling is dispensable in the development of autoimmune type 1 diabetes in NOD mice.
There have been two previous conflicting reports that the development of T-cell-mediated autoimmune diabetes (type 1 diabetes) was respectively unaffected or inhibited in NOD mice genetically deficient in the T-helper (Th) 1 cytokine interferon (IFN)-gamma or the alpha-chain subunit of its receptor. Our goal was to resolve this conundrum by congenically transferring, from a 129 donor strain to the NOD background, a functionally inactivated gene for the beta-chain signaling (located on chromosome 16) rather than the alpha-chain ligand binding domain (located on chromosome 10) of the IFN-gamma receptor. These NOD.IFNgammaRBnull mice were characterized by normal patterns of leukocyte development and T-cells that produced greatly enhanced levels of the putatively type 1 diabetes-protective Th2 cytokine interleukin (IL)-4. However, despite being unable to respond to the primary Thl cytokine IFN-gamma and having T-cells that produce greatly enhanced levels of IL-4, NOD.IFNgammaRBnull mice remained highly susceptible to type 1 diabetes. This result indicated that the previously reported inhibition of type 1 diabetes in NOD mice carrying a functionally inactivated IFN-gamma receptor alpha-chain gene may have been due to a closely linked and previously unidentified diabetes resistance allele. Furthermore, our results indicate that the pathogenicity of diabetogenic T-cells in NOD mice is not dampened by an inability to respond to IFN-gamma and a concurrent shift to greatly enhanced Th2 cytokine production. This finding calls into question whether clinical protocols designed to shift beta-cell autoreactive T-cells from a Thl to Th2 cytokine production profile will truly be safe and efficacious in blocking the development of type 1 diabetes in humans.


Methylene blue prevents surgery-induced peritoneal adhesions but impairs the early phase of anastomotic wound healing.
Adhesion formation continues to be an important problem in gastrointestinal surgery. In recent years, methylene blue (MB) has been reported to be an effective agent for preventing peritoneal adhesions. However, its effects on the wound healing process are unknown. In the present study, we investigated the effects of MB on the early and late phases of anastomotic wound healing and on adhesion formation. We randomly categorized 92 rats into 2 groups in bursting pressure measurements and 50 rats into 3 groups in the adhesion model. We divided the animals into saline-treated (n = 46) or MB-treated (n = 46) groups. Bursting pressures of the anastomoses were measured on postoperative days 3 and 7. In biochemical studies, tissue hydroxyproline levels, total nitrite/nitrate levels and nitric oxide synthase activity were measured on postoperative days 3 and 7. In the adhesion model, we randomly categorized rats into sham (n = 10), saline-treated (n = 20) and MB-treated (n = 20) groups, and the formation of intraperitoneal adhesions was scored on postoperative day 14. We compared the measurement of bursting pressure and biochemical measurements of tissue hydroxyproline levels, total nitrite/nitrate levels and nitric oxide synthase activity. Histopathological findings of specimens were presented. During the early phase of wound healing (postoperative day 3), bursting pressures, tissue hydroxyproline, total nitrite/nitrate levels and nitric oxide synthase activity in the MB-treated group were significantly lower than those of the saline-treated group. On postoperative day 7, there was no significant difference in these parameters between MB and saline-treated groups. In the adhesion model, MB caused a significant reduction in the formation of peritoneal adhesions. MB prevents peritoneal adhesions but causes a significant impairment of anastomotic bursting pressure during the early phase of the wound healing process by its transient inhibitory effect on the nitric oxide pathway.


Neurophysiological evidence for the country-of-origin effect: an event-related potential study.
Consumers often rely on observable cues that hint at the hidden quality of a product. The aim of this study was to investigate brain activities associated with the country-of-origin (COO) effect and consumer evaluation of a product design. Electroencephalogram recordings were used to observe event-related brain potentials associated with the COO effect and design evaluation. We found that the frontocentral N90 and parieto-occipital P220 amplitudes are involved in forming preference to design, whereas the COO effect is processed in the centroparietal P500 amplitude. We also found a significant interaction effect between COO and design preference with regard to reaction times. Specifically, participants tended to spend more time making a preference decision when they did not like the product design made in a country with a favorable COO. These results imply that the two cognitive processes, evaluation of COO and formation of design preference, are activated independently at an early stage. It also suggests that these two processes interact with each other toward the end of the decision phase. Together, the results of this study provide neuropsychological evidence supporting a significant role of COO in the formation of design preference. Future studies are required to further delve into other neurophysiological activities associated with the COO effect.


A mass-spring model unveils the morphogenesis of phototrophic Diatoma biofilms.
Diatoms often dominate planktonic communities in the ocean and phototrophic biofilms in streams and rivers, greatly contributing to global biogeochemical fluxes. In pelagic ecosystems, these microscopic algae can form chain-like microcolonies, which seem advantageous for nutrient uptake and protect against grazing, and at the same time reduce sinking. Despite the capability of many diatoms to form chains, their contribution to the architecture of phototrophic biofilms remains elusive. Here we propose a computational model to simulate the growth and behaviour of Diatoma chains in contrasting flow environments. This mass-spring mechanical model captures the natural behaviour of Diatoma chains well, emphasising the relevance of chain growth and entanglement for biofilm morphogenesis. The model qualitatively describes formation of intricate dome-shaped structures and of dreadlock-type streamers as observed in nature in multidirectional and unidirectional flow, respectively. The proposed model is a useful tool to study the effect of fluid dynamics on biofilm morphogenesis.


SOX9 accelerates ESC differentiation to three germ layer lineages by repressing SOX2 expression through P21 (WAF1/CIP1).
Upon removal of culture conditions that maintain an undifferentiated state, mouse embryonic stem cells (ESCs) differentiate into various cell types. Differentiation can be facilitated by forced expression of certain transcription factors (TFs), each of which can generally specify a particular developmental lineage. We previously established 137 mouse ESC lines, each of which carried a doxycycline-controllable TF. Among them, Sox9 has unique capacity: its forced expression accelerates differentiation of mouse ESCs into cells of all three germ layers. With the additional use of specific culture conditions, overexpression of Sox9 facilitated the generation of endothelial cells, hepatocytes and neurons from ESCs. Furthermore, Sox9 action increases formation of p21 (WAF1/CIP1), which then binds to the SRR2 enhancer of pluripotency marker Sox2 and inhibits its expression. Knockdown of p21 abolishes inhibition of Sox2 and Sox9-accelerated differentiation, and reduction of Sox2 2 days after the beginning of ESC differentiation can comparably accelerate mouse ESC formation of cells of three germ layers. These data implicate the involvement of the p21-Sox2 pathway in the mechanism of accelerated ESC differentiation by Sox9 overexpression. The molecular cascade could be among the first steps to program ESC differentiation.


Use of Truquant BR radioimmunoassay for early detection of breast cancer recurrence in patients with stage II and stage III disease.
The Truquant BR radioimmunoassay (RIA) (Biomira Diagnostics Inc, Rexdale, Canada) uses the monoclonal antibody B27.29 to quantitate the MUC-1 gene product (CA 27.29 antigen) in serum. We evaluated CA 27.29 antigen in a controlled, prospective clinical trial for its ability to predict relapse in stage II and stage III breast cancer patients. Over a 2-year period, 166 patients who had completed therapy for stage II (80.1%) or III (19.9%) breast cancer and were clinically free of disease were serially tested for CA 27.29 antigen levels. The study was double-masked and cancer recurrence was documented based on clinical findings. Patients with two consecutive CA 27.29 antigen test results above the upper limit of normal were considered positive. The Truquant BR RIA had a sensitivity of 57.7%, specificity of 97.9%, positive predictive value of 83.3%, and negative predictive value of 92.6%. The recurrence rate was 15.7%. A Cox regression analysis showed that the only variable to correlate with recurrent disease was the CA 27.29 antigen test result. Patients with a positive test result had increased odds of having a recurrence (odds ratio, 6.8; P < .00001). The test was effective in predicting recurrence in patients with both distant and locoregional disease. In a subgroup of patients with bone pain, CA 27.29 antigen level was found to identify reliably patients who would subsequently develop recurrent disease. These data demonstrate that the Truquant BR RIA can be used as an aid to predict recurrent breast cancer in patients with stage II and III disease.


p53 protein expression independently predicts outcome in patients with lower-risk myelodysplastic syndromes with del(5q).
Del(5q) myelodysplastic syndromes defined by the International Prognostic Scoring System as low- or intermediate-1-risk (lower-risk) are considered to have an indolent course; however, recent data have identified a subgroup of these patients with more aggressive disease and poorer outcomes. Using deep sequencing technology, we previously demonstrated that 18% of patients with lower-risk del(5q) myelodysplastic syndromes carry TP53 mutated subclones rendering them at higher risk of progression. In this study, bone marrow biopsies from 85 patients treated with lenalidomide in the MDS-004 clinical trial were retrospectively assessed for p53 expression by immunohistochemistry in association with outcome. Strong p53 expression in ≥ 1% of bone marrow progenitor cells, observed in 35% (30 of 85) of patients, was significantly associated with higher acute myeloid leukemia risk (P=0.0006), shorter overall survival (P=0.0175), and a lower cytogenetic response rate (P=0.009), but not with achievement or duration of 26-week transfusion independence response. In a multivariate analysis, p53-positive immunohistochemistry was the strongest independent predictor of transformation to acute myeloid leukemia (P=0.0035). Pyrosequencing analysis of laser-microdissected cells with strong p53 expression confirmed the TP53 mutation, whereas cells with moderate expression predominantly had wild-type p53. This study validates p53 immunohistochemistry as a strong and clinically useful predictive tool in patients with lower-risk del(5q) myelodysplastic syndromes. This study was based on data from the MDS 004 trial (clinicaltrials.gov identifier: NCT00179621).


Neural-vascular relationships in central retina of macaque monkeys (Macaca fascicularis).
The relationship of the vasculature to the neuronal layers was studied in whole-mounts and in sections of macaque retinas. Like other central nervous structures, primate retinas have local variations in vascularity that reflect local variations in metabolism, rather than simply tissue thickness or volume. A special feature of the retina is a dense vascular plexus in the nerve fiber layer, which is unmyelinated and hence must generate a substantial metabolic demand for ion pumping. Much of the retinal vasculature is laminated and located at specific layer boundaries. Throughout the central retina, two planes of capillaries bracket the inner nuclear layer to form a sclerad capillary network. In some regions, especially near the fovea, a second, more vitread network brackets the ganglion cell layer with another pair of capillary planes. Wherever the nerve fiber layer is thick, the vitread network becomes less planar and is multilayered. When surrounded by nerve fibers, capillaries tend to orient parallel to the fibers; when adjacent to ganglion cell bodies, the capillaries are less systematically oriented. At the border between the nerve fiber layer and the ganglion cell layer, rows of ganglion cells often interdigitate with nerve fiber bundles, resulting in local perturbations of capillary orientation. The volume of the sclerad capillary network is relatively constant at different locations, but the volume of the vitread network increases dramatically where the nerve fiber layer is thick. As a result, the vascularity of the retina is greatest in the peripapillary region near the optic disk, even though the total thickness of the peripapillary retina is comparable to the retinal thickness near the foveal crest. As many as 60-70% of the photons passing through the retina in the peripapillary region will encounter one or more capillaries before reaching a photoreceptor. Median capillary diameter increases with retinal depth from 4.5-4.7 microns in the nerve fiber layer to 5.0 microns at the sclerad border of the inner nuclear layer. Capillary diameter in the nerve fiber layer also increases near the optic disk.


Response to MLN8237 in pancreatic cancer is not dependent on RalA phosphorylation.
The high prevalence of KRAS mutations and importance of the RalGEF-Ral pathway downstream of activated K-ras in pancreatic ductal adenocarcinoma (PDAC) emphasize the importance of identifying novel methods by which to therapeutically target these pathways. It was recently demonstrated that phosphorylation of RalA S194 by Aurora A kinase (AAK) is critical for PDAC tumorigenesis. We sought to evaluate the AAK-selective inhibitor MLN8237 as a potential indirect anti-RalA-targeted therapy for PDAC. We used a site-specific phospho-S194 RalA antibody and determined that RalA S194 phosphorylation levels were elevated in a subset of PDAC cell lines and human tumors relative to unmatched normal controls. Effects of MLN8237 on anchorage-independent growth in PDAC cell lines and growth of patient-derived xenografts (PDX) were variable, with a subset of cell lines and PDX showing sensitivity. Surprisingly, RalA S194 phosphorylation levels in PDAC cell lines or PDX tumors did not correlate with MLN8237 responsiveness. However, we identified Ki67 as a possible early predictive biomarker for response to MLN8237 in PDAC. These results indicate that MLN8237 treatment may be effective for a subset of patients with PDAC independent of RalA S194 phosphorylation. Ki67 may be an effective pharmacodynamic biomarker to identify response early in the course of treatment.


Dengue virus or NS1 protein induces trans-endothelial cell permeability associated with VE-Cadherin and RhoA phosphorylation in HMEC-1 cells preventable by Angiopoietin-1.
A transient increase in trans-endothelial cell permeability in dengue patients leads to vascular leakage and shock syndrome. Here, we analysed the molecular mechanisms that cause permeability changes in human dermal microvascular endothelial cells (HMEC-1) using a direct dengue virus (DENV) infection model or treatment with NS1, a secreted DENV non-structural protein. In HMEC-1 cells, both treatments increase permeability with a concordant increase in the secretion of angiopoietin-2 (Ang-2). There is phosphorylation and loss of the junction protein VE-Cadherin from the inter-endothelial cell junctions and phosphorylation of RhoA. Direct virus infection results in activation of Src by phosphorylation, whereas NS1 treatment alone does not lead to Src activation. Furthermore, treatment with recombinant Ang-1, a physiological antagonist of Ang-2, prevents Ang-2 release, VE-Cadherin phosphorylation and internalization, and phosphorylation of RhoA and Src, resulting in restoration of barrier function. The permeability increase could also be prevented by blocking the Ang1/2 signalling receptor, Tie-2, or using a Rho/ROCK-specific inhibitor. Dasatinib, a Src-family kinase (SFK) inhibitor that inhibits Src phosphorylation, prevents enhanced permeability induced by direct DENV infection whereas in NS1 protein-treated cells its effect is less significant. The results provide important insights on the mechanisms of increased trans-endothelial permeability in DENV infection, and suggest the therapeutic potential of using recombinant Ang-1 or targeting these key molecules to prevent vascular leakage in dengue.


Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice.
The N-terminal 17 amino acids of huntingtin (NT17) can be phosphorylated on serines 13 and 16; however, the significance of these modifications in Huntington's disease pathogenesis remains unknown. In this study, we developed BAC transgenic mice expressing full-length mutant huntingtin (fl-mhtt) with serines 13 and 16 mutated to either aspartate (phosphomimetic or SD) or alanine (phosphoresistant or SA). Both mutant proteins preserve the essential function of huntingtin in rescuing knockout mouse phenotypes. However, fl-mhtt-induced disease pathogenesis, including motor and psychiatric-like behavioral deficits, mhtt aggregation, and selective neurodegeneration are abolished in SD but preserved in SA mice. Moreover, modification of these serines in expanded repeat huntingtin peptides modulates aggregation and amyloid fibril formation in vitro. Together, our findings demonstrate that serines 13 and 16 are critical determinants of fl-mhtt-induced disease pathogenesis in vivo, supporting the targeting of huntingtin NT17 domain and its modifications in HD therapy.


Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.
The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.


Results of the GEP-ISFG collaborative study on the Y chromosome STRs GATA A10, GATA C4, GATA H4, DYS437, DYS438, DYS439, DYS460 and DYS461: population data.
The Spanish and Portuguese ISFG Working Group (GEP-ISFG) carried out a collaborative exercise in order to asses the performance of two Y chromosome STR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The groups that reported correct results in all the systems were also asked to analyse a population sample in order to evaluate the informative content of these STRs in different populations. A total of 1020 males out of 13 population samples from Argentina, Brazil, Costa Rica, Macao, Mozambique, Portugal and Spain were analysed for all the loci included in the present study. Haplotype and allele frequencies of these eight Y-STRs were estimated in all samples. The lowest haplotype diversity was found in the Lara (Argentina) population (95.44%) and the highest (99.90%) in Macao (China). Pairwise haplotype analysis showed the relative homogeneity of the Iberian origin samples, in accordance with what was previously found in the European populations for other Y-STR haplotypes (http://www.ystr.org). As expected, the four non-Caucasian samples, Macao (Chinese), Mozambique (Africans), Costa Rica (Africans) and Argentina (Lara, Amerindians), show highly significant Phist values in the pairwise comparisons with all the Caucasian samples.


Geometry of abstract learned knowledge in the hippocampus.
Hippocampal neurons encode physical variables1-7 such as space1 or auditory frequency6 in cognitive maps8. In addition, functional magnetic resonance imaging studies in humans have shown that the hippocampus can also encode more abstract, learned variables9-11. However, their integration into existing neural representations of physical variables12,13 is unknown. Here, using two-photon calcium imaging, we show that individual neurons in the dorsal hippocampus jointly encode accumulated evidence with spatial position in mice performing a decision-making task in virtual reality14-16. Nonlinear dimensionality reduction13 showed that population activity was well-described by approximately four to six latent variables, which suggests that neural activity is constrained to a low-dimensional manifold. Within this low-dimensional space, both physical and abstract variables were jointly mapped in an orderly manner, creating a geometric representation that we show is similar across mice. The existence of conjoined cognitive maps suggests that the hippocampus performs a general computation-the creation of task-specific low-dimensional manifolds that contain a geometric representation of learned knowledge.


Mathematical description of pH-stat kinetic traces measured during photochemical quinone decomposition.
Substituted 1,4-benzoquinone (QR) derivatives are photosensitive in aqueous solution and form hydroquinones (QR-H2) and hydroxy-quinones (QR-OH), two weak acids in their photoreaction. For this reason, the kinetics of the photoreaction can be conveniently followed by the pH-stat titration technique. The mathematical description of the kinetic traces measured provides the two main parameters of the photoreaction: the differential quantum yield of the reaction (Φ) and the ratio of the two photo-products, i.e. the fraction of QR that is converted to QR-OH (α). These values are described in this paper for 2,5-dichloro-1,4-benzoquinone at different pH values, together with the detailed mathematical evaluation of the application limits of the pH-stat method for such reactions.


The N-terminal adenosine triphosphate binding domain of Hsp90 is necessary and sufficient for interaction with estrogen receptor.
To understand how the molecular chaperone Hsp90 participates in conformational maturation of the estrogen receptor (ER), we analyzed the interaction of immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was either immunoadsorbed to BF4 antibody-Sepharose or GST-Hsp90 fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain specifically ER, similarly to immunoadsorbed Hsp90. When cells were treated with estradiol and the hormone treatment was maintained during cell homogenization, binding, and washing steps, GST.90 still interacted efficiently with ER, suggesting that ER may form complexes with Hsp90 even after its activation by hormone and salt extraction from nuclei. The GST.90-ER interaction was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously stabilized by molybdate, indicating that GST.90-ER complexes behave like cytosolic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other chaperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1-224), the interaction being more efficient when the charged region A was present in the mutant (1-334). The N-terminal fragment 1-334, devoid of the dimeric GST moiety, was also able to interact with ER, pointing to the monomeric N-terminal adenosine triphosphate binding region of Hsp90 (1-224) as the region necessary and sufficient for interaction. These results contribute to understand the Hsp90-dependent process responsible for conformational competence of ER.


OmpT: molecular dynamics simulations of an outer membrane enzyme.
Five molecular dynamics simulations (total duration >25 ns) have been performed on the Escherichia coli outer membrane protease OmpT embedded in a dimyristoylphosphatidylcholine lipid bilayer. Globally the protein is conformationally stable. Some degree of tilt of the beta-barrel is observed relative to the bilayer plane. The greatest degree of conformational flexibility is seen in the extracellular loops. A complex network of fluctuating H-bonds is formed between the active site residues, such that the Asp210-His212 interaction is maintained throughout, whereas His212 and Asp83 are often bridged by a water molecule. This supports a catalytic mechanism whereby Asp83 and His212 bind a water molecule that attacks the peptide carbonyl. A configuration yielded by docking calculations of OmpT simulation snapshots and a model substrate peptide Ala-Arg-Arg-Ala was used as the starting point for an extended Huckel calculation on the docked peptide. These placed the lowest unoccupied molecular orbital mainly on the carbon atom of the central C=O in the scissile peptide bond, thus favoring attack on the central peptide by the water held by residues Asp83 and His212. The trajectories of water molecules reveal exchange of waters between the intracellular face of the membrane and the interior of the barrel but no exchange at the extracellular mouth. This suggests that the pore-like region in the center of OmpT may enable access of water to the active site from below. The simulations appear to reveal the presence of specific lipid interaction sites on the surface of the OmpT barrel. This reveals the ability of extended MD simulations to provide meaningful information on protein-lipid interactions.


Physical Function in an Aging Population in Rural South Africa: Findings From HAALSI and Cross-National Comparisons With HRS Sister Studies.
We use recently-collected data from the Health and Aging in Africa: a Longitudinal Study of an INDEPTH Community in South Africa (HAALSI) cohort from Agincourt, South Africa, to describe physical functioning in this aging population, and place the overall level and age-trajectories of physical health in the context of other Health and Retirement Study (HRS) sister studies in low- and middle-income countries (LMICs). We conduct multiple regression to estimate associations of physical functioning assessed from both self-report (activities of daily living [ADL] limitation, self-reported health) and performance (grip strength, gait speed) with socio-demographic and health characteristics in HAALSI, and use fully-interacted regression models to compare age-patterns of physical functioning outcomes cross-nationally. Gender differences in self-reported health are minimal, and men had 30% higher odds of being ADL limited controlling for socio-demographic and health characteristics. Measured physical performance is closely tied with socioeconomic conditions, but self-reported measures have a much smaller or weaker socioeconomic gradient. In international age-adjusted comparisons, the HAALSI sample had lower physical performance outcomes than most comparison populations. As the first HRS sister study undertaken in Africa, HAALSI adds vital information on population aging and health in the region. Continuing waves of HAALSI data will be a key resource for understanding differences in the complex processes of disability across LMIC contexts.


Knowledge and attitude towards organ donation among adult population in Al-Kharj, Saudi Arabia.
Organ transplantation is a lifesaving treatment for patients with end-stage organ failure. Despite the advanced medical science and technology, shortage of organs had led to a growing gap between the demand for organs and the number of donors. With a limited number of studies on the subject and based on those findings, the public knowledge and attitudes must be assessed to understand more clearly that why many people are opposing donating their organs in Saudi Arabia. The objective of our study was to assess the knowledge and attitude of the adult population toward organ donation in Saudi Arabia. This was a hospital-based cross-sectional study where the information was collected using a self-administered questionnaire in Al-Kharj, Saudi Arabia. The questionnaire was distributed in both King Khalid Hospital and Prince Sattam Bin Abdulaziz University Hospital, and data gathered analyzed by Statistical Package for Social Sciences (SPSS version 20.0). There were a total of 403 respondents. Nearly 35.6% did not have the knowledge that organ donation is legal in the KSA. Almost 97% did not know where to go if they want to become donors. All of who were willing to donate, the most common reason was to save someone's life (92.7%). Body distortion (39%) and fear of health complications (35%) were the most common causes people opposed donation. It was suggested that, in order to increase the awareness for organ donation, the important role of health workers and hospital displays should be immediately addressed and public lectures should be held on regular basis. Information regarding organ donation should be incorporated with clear messages in various mass media.


An 'In the Wild' Experiment on Presence and Embodiment using Consumer Virtual Reality Equipment.
Consumer virtual reality systems are now becoming widely available. We report on a study on presence and embodiment within virtual reality that was conducted 'in the wild', in that data was collected from devices owned by consumers in uncontrolled settings, not in a traditional laboratory setting. Users of Samsung Gear VR and Google Cardboard devices were invited by web pages and email invitation to download and run an app that presented a scenario where the participant would sit in a bar watching a singer. Each participant saw one of eight variations of the scenario: with or without a self-avatar; singer inviting the participant to tap along or not; singer looking at the participant or not. Despite the uncontrolled situation of the experiment, results from an in-app questionnaire showed tentative evidence that a self-avatar had a positive effect on self-report of presence and embodiment, and that the singer inviting the participant to tap along had a negative effect on self-report of embodiment. We discuss the limitations of the study and the platforms, and the potential for future open virtual reality experiments.


Placental Villous Vascularity Is Decreased in Premature Infants with Bronchopulmonary Dysplasia-Associated Pulmonary Hypertension.
The development of pulmonary hypertension (PH) is a serious complication of bronchopulmonary dysplasia (BPD) among infants born at extremely low gestational ages. Bronchopulmonary dysplasia-associated PH is characterized by persistent pulmonary vasoconstriction, progressive right heart dysfunction, and an increased risk of death. We have shown previously that certain placental vascular lesions are associated with BPD-associated PH. Further evaluation of the villous and vascular morphometry of these placentas is warranted. Using digital image analysis (DIA), we compared villous and vascular morphometric parameters of placentas from infants with and without BPD-associated PH. We conducted a case-control study of placentas from 14 infants born at ≤28 weeks' gestational age (GA). Cases with PH (N=7) and non-PH controls (N=7) were identified using echocardiogram screening at 36 weeks' corrected GA. Central parenchymal sections from each placenta were stained for CD31. Digital image analysis was used to measure vessel and villous capillary number, perimeter, diameter, and area. Mean villous vascularity (number of vessels per villus) was calculated for each patient. Mean vessel and villous number as well as area were similar between the two groups. Villous vascularity was decreased in placentas from infants who ultimately had PH disease compared to non-PH controls (5.5±1.0 vs 7.1±1.6; P<0.05). Placental villous vascularity is decreased in infants with BPD-associated PH. Further studies should assess whether placental morphometric markers may allow clinicians to better predict BPD and provide earlier and more targeted management.


Combined immune thrombocytopenic purpura and immunoglobulin A nephropathy: a similar pathophysiologic process?
IgA nephropathy is one of the most common forms of glomerulopathies. It is an immune complex-mediated glomerulonephritis diagnosed by the presence of mesangial IgA deposits that are often associated with mesangial cell proliferation. The IgG, C3, IgM, or other immunoglobulin light chains may be co-existed with IgA. Its pathogenesis suggested that it is responsible for enhancing the production of proinflammatory cytokines, chemokines, and growth factors. Platelet-derived growth factor (PDGF) has also been implicated as a modulator of disease activity. Immune thrombocytopenic purpura (ITP) is a bleeding disorder caused by thrombocytopenia that is not associated with a systemic disease. Its pathogenesis suggested an autoimmune disease in which IgG is thought to damage megakaryocytes, which are the precursors of platelet cells. Several studies reported that PDGF levels were higher in normal subjects than in patients with ITP. Moreover, ITP is a disease related to the antibody. Thus, our aim is to examine whether a similar pathophysiological relationship exist between ITP and IgAN that may be mediated by PDGF and/or IgG.


Predicting protein-protein interaction by searching evolutionary tree automorphism space.
Uncovering the protein-protein interaction network is a fundamental step in the quest to understand the molecular machinery of a cell. This motivates the search for efficient computational methods for predicting such interactions. Among the available predictors are those that are based on the co-evolution hypothesis "evolutionary trees of protein families (that are known to interact) are expected to have similar topologies". Many of these methods are limited by the fact that they can handle only a small number of protein sequences. Also, details on evolutionary tree topology are missing as they use similarity matrices in lieu of the trees. We introduce MORPH, a new algorithm for predicting protein interaction partners between members of two protein families that are known to interact. Our approach can also be seen as a new method for searching the best superposition of the corresponding evolutionary trees based on tree automorphism group. We discuss relevant facts related to the predictability of protein-protein interaction based on their co-evolution. When compared with related computational approaches, our method reduces the search space by approximately 3 x 10(5)-fold and at the same time increases the accuracy of predicting correct binding partners.


Modelling the effect of market forces on the impact of introducing human immunodeficiency virus pre-exposure prophylaxis among female sex workers.
Pre-exposure prophylaxis (PrEP) to prevent human immunodeficiency virus (HIV) enables female sex workers (FSWs) to protect themselves from HIV without relying on clients using condoms. Yet, because PrEP reduces HIV risk, financial incentives to not use condoms may lead to risk compensation: reductions in condom use and/or increases in commercial sex, and may reduce the price of unprotected sex. In this analysis, we integrate market forces into a dynamic HIV transmission model to assess how risk compensation could change the impact of PrEP among FSWs and clients. We parameterise how sexual behavior may change with PrEP use among FSWs using stated preference data combined with economic theory. Our projections suggest the impact of PrEP is sensitive to risk compensatory behaviors driven by changes in the economics of sex work. Condom substitution could reduce the impact of PrEP on HIV incidence by 55%, while increases in the frequency of commercial sex to counter decreases in the price charged for unprotected sex among PrEP users could entirely mitigate the impact of PrEP. Accounting for competition between PrEP users and nonusers exacerbates this further. Alternative scenarios where increases in unprotected sex among PrEP users are balanced by decreases in non-PrEP users have the opposite effect, resulting in PrEP having much greater impact. Intervention studies need to determine how HIV prevention products may change the economics of sex work and provision of unprotected sex to enable a better understanding of their impact.


Elucidation of chemical modifier reactivity towards peptides and proteins and the analysis of specific fragmentation by matrix-assisted laser desorption/ionization collision-induced dissociation tandem mass spectrometry.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of covalent 5-lipoxygenase inhibitors is challenging due to unknown amino acid specificity and low posttranslational modification (PTM)-identification rates. The analysis of the amino-acid specificity and of the characteristic fragmentation of chemically modified peptides is considered to improve knowledge for the analysis of chemically modified peptides and proteins by MALDI-MS. Various compounds were used to investigate the modification of synthetic peptides carrying reactive amino acid residues. Mass spectra were recorded using a MALDI-LTQ Orbitrap XL for high-resolution mass spectrometry and ion trap MALDI-MS2 . UV-Vis-based reduction and radical scavenging analysis was conducted. The on-plate digestion method described by Rühl et al was utilized for modification-site analysis at 5-lipoxygenase. The analysis of amino-acid-specific reactivity revealed the reactivity of quinones towards cysteine residues and the potential occurrence of a subsequent oxidative process was observed by an UV-Vis-based reduction assay. MALDI collision-induced dissociation tandem mass spectrometry (CID-MS2 ) indicated a prominent fragmentation mechanism of modified cysteine and histidine residues. Fragmentation included highly abundant neutral-loss signals which could be used to identify new modifications induced by chemical modifiers at the cysteine-159 residue of 5-lipoxygenase. Specificity and fragmentation analysis provides crucial information for the analysis of chemically modified cysteines and histidines by MALDI-MS. Elucidation of binding sites by MALDI-MS has been significantly improved using an easy-to-run peptide assay and gives background information for the analysis in the case of chemically modified 5-lipoxygenase.


Triheptanoin: A Rescue Therapy for Cardiogenic Shock in Carnitine-acylcarnitine Translocase Deficiency.
Carnitine-acylcarnitine translocase (CACT) deficiency is a rare long-chain fatty acid oxidation disorder (LC-FAOD) with high mortality due to cardiomyopathy or lethal arrhythmia. Triheptanoin (UX007), an investigational drug composed of synthetic medium odd-chain triglycerides, is a novel therapy in development for LC-FAOD patients. However, cases of its safe and efficacious use to reverse severe heart failure in CACT deficiency are limited. Here, we present a detailed report of an infant with CACT deficiency admitted in metabolic crisis that progressed into severe cardiogenic shock who was successfully treated by triheptanoin. The child was managed, thereafter, on triheptanoin until her death at 3 years of age from a cardiopulmonary arrest in the setting of acute respiratory illness superimposed on chronic hypercarbic respiratory failure.


Glibenclamide treatment in a Cantú syndrome patient with a pathogenic ABCC9 gain-of-function variant: Initial experience.
Cantú syndrome (CS), characterized by hypertrichosis, distinctive facial features, and complex cardiovascular abnormalities, is caused by pathogenic variants in ABCC9 and KCNJ8 genes. These genes encode gain-of-function mutations in the regulatory (SUR2) and pore-forming (Kir6.1) subunits of KATP channels, respectively, suggesting that channel-blocking sulfonylureas could be a viable therapy. Here we report a neonate with CS, carrying a heterozygous ABCC9 variant (c.3347G>A, p.Arg1116His), born prematurely at 32 weeks gestation. Initial echocardiogram revealed a large patent ductus arteriosus (PDA), and high pulmonary pressures with enlarged right ventricle. He initially received surfactant and continuous positive airway pressure ventilation and was invasively ventilated for 4 weeks, until PDA ligation. After surgery, he still had ongoing bilevel positive airway pressure (BiPAP) requirement, but was subsequently weaned to nocturnal BiPAP. He was treated for pulmonary hypertension with Sildenafil, but failed to make further clinical improvement. A therapeutic glibenclamide trial was commenced in week 11 (initial dose of 0.05 mg-1 kg-1 day-1 in two divided doses). After 1 week of treatment, he began to tolerate time off BiPAP when awake, and edema improved. Glibenclamide was well tolerated, and the dose was slowly increased to 0.15 mg-1 kg-1 day-1 over the next 12 weeks. Mild transient hypoglycemia was observed, but there was no cardiovascular dysfunction. Confirmation of therapeutic benefit will require studies of more CS patients but, based on this limited experience, consideration should be given to glibenclamide as CS therapy, although problems associated with prematurity, and complications of hypoglycemia, might limit outcome in critically ill neonates with CS.


Introduction: Childhood implications of parental aging.
Men and women are increasingly delaying childbearing to the late 30s, the 40s and beyond. The implications of this societal change on childhood health and well-being have only recently been a focus of research. There are known increased perinatal risks associated with increasing maternal age, while paternal age seems to have a potentially greater negative impact on childhood health. Understanding the mechanisms underlying the aging of sperm and eggs, and how these changes impact offspring, is a critical next step as we work to help patients build healthy families.


Food Intolerance and childhood asthma: what is the link?
Food allergies and asthma are increasing worldwide. It is estimated that approximately 8% of children aged <3 years have food allergies. Foods can induce a variety of IgE-mediated, cutaneous, gastrointestinal, and respiratory reactions. The most common foods responsible for allergic reactions in children are egg, milk, peanut, soy, fish, shellfish, and tree nuts. Asthma alone as a manifestation of a food allergy is rare and atypical. Less than 5% of patients experience wheezing without cutaneous or gastrointestinal symptoms during a food challenge. In addition to acute respiratory symptoms, a food allergy may also induce airway hyper-responsiveness beyond the initial reaction. This process can occur in patients who do not demonstrate a decrease in lung function during the reaction. Inhalation of aerosolized food particles can cause respiratory symptoms in selected food-allergic individuals, particularly with fish and shellfish during cooking and aerosolization. However, this has not been demonstrated with the smelling of, or casual contact with, peanut butter. Rarely, food additives such as sulfating agents can cause respiratory reactions. This reaction occurs primarily in patients with underlying asthma, particularly in patients with more severe asthma. In contrast, there is no convincing evidence that tartrazine or monosodium glutamate can induce asthma responses. Although food-induced asthma is rare, it is common for patients and clinicians to perceive that food can trigger asthma. Avoidance of specific foods or additives has not been shown to improve asthma, even in patients who may perceive that a particular food worsens their asthma.However, patients with underlying asthma are more likely to experience a fatal or near-fatal food reaction. Food reactions tend to be more severe or life threatening when they involve the respiratory tract. The presence of a food allergy is a risk factor for the future development of asthma, particularly for children with sensitization to egg protein. The diagnosis of a food allergy includes skin or in vitro testing as an initial study when the history suggests food allergy. While negative testing generally rules out a food allergy, positive testing should be followed by a food-challenge procedure for a definitive diagnosis. The CAP-RAST FEIA (CAP-radioallergosorbent test [RAST] fluoroenzyme immunoasssay system [FEIA]) is an improved in vitro measure that in some cases may decrease the need for food challenges. However, similar to skin testing and the RAST, there is good sensitivity but poor specificity, such that specific challenges are often warranted.


Interhemispheric pathways of the hippocampal formation, presubiculum, and entorhinal and posterior parahippocampal cortices in the rhesus monkey: the structure and organization of the hippocampal commissures.
The interhemispheric pathways originating in the hippocampal formation, presubiculum, and entorhinal and posterior parahippocampal cortices and coursing through the fornix system were investigated by autoradiographic tracing in 29 rhesus monkeys (Macaca mulatta). The results revealed that crossing fibers are segregated into three contiguous systems. A ventral hippocampal commissure lies at the transition between the body and anterior columns of the fornix in the vicinity of the subfornical organ and the interventricular foramina of Monro; it is formed by axons arising in the most anterior (uncal and genual) subdivisions of the hippocampal formation. A dorsal hippocampal commissure lies inferior to the posterior end of the body of the corpus callosum; it is formed by axons arising in the presubiculum and entorhinal cortex of the anterior parahippocampal gyrus and the proisocortical and neocortical subdivisions of the posterior parahippocampal gyrus but not in the hippocampal formation. A hippocampal decussation lies between the ventral hippocampal commissure and dorsal hippocampal commissure; it is formed by axons arising in the body of the hippocampal formation. In contrast to the fibers of the ventral hippocampal commissure and dorsal hippocampal commissure, which terminate in contralateral cortical areas, these decussating fibers terminate in the contralateral septum. Thus, the ventral hippocampal commissure and dorsal hippocampal commissure of the rhesus monkey appear to be homologous to similarly designated structures in other mammals. To the extent that these observations also apply to the interhemispheric fibers of the human hippocampal formation and parahippocampal areas, their possible preservation must be considered when interpreting the effect of callosal transection on seizures and the results of "split-brain" studies, since callosal transection may fail to sever the hippocampal commissures in their entirety.


A new reference genome for Sorghum bicolor reveals high levels of sequence similarity between sweet and grain genotypes: implications for the genetics of sugar metabolism.
The process of crop domestication often consists of two stages: initial domestication, where the wild species is first cultivated by humans, followed by diversification, when the domesticated species are subsequently adapted to more environments and specialized uses. Selective pressure to increase sugar accumulation in certain varieties of the cereal crop Sorghum bicolor is an excellent example of the latter; this has resulted in pronounced phenotypic divergence between sweet and grain-type sorghums, but the genetic mechanisms underlying these differences remain poorly understood. Here we present a new reference genome based on an archetypal sweet sorghum line and compare it to the current grain sorghum reference, revealing a high rate of nonsynonymous and potential loss of function mutations, but few changes in gene content or overall genome structure. We also use comparative transcriptomics to highlight changes in gene expression correlated with high stalk sugar content and show that changes in the activity and possibly localization of transporters, along with the timing of sugar metabolism play a critical role in the sweet phenotype. The high level of genomic similarity between sweet and grain sorghum reflects their historical relatedness, rather than their current phenotypic differences, but we find key changes in signaling molecules and transcriptional regulators that represent new candidates for understanding and improving sugar metabolism in this important crop.


Ethnicity and nonalcoholic fatty liver disease in an obesity clinic: the impact of triglycerides.
Nonalcoholic fatty liver disease (NAFLD) is a growing problem that is associated with the metabolic syndrome. The goal of the present study was to evaluate for ethnic differences in NAFLD and clinical correlates of NAFLD. The study population consisted of 567 patients seen at an urban obesity clinic. Elevated aminotransferase levels were used as a surrogate marker for NAFLD. The prevalence of elevated aminotransferases was highest in Hispanics (39%), followed by Caucasians (28%), and African Americans (12%). In univariate analysis, elevated aminotransferase levels were associated with ethnicity (Hispanic > African American, P < 0.001, and Caucasian > African American P = 0.030), hypertriglyceridemia (P < 0.001), and male gender (P < 0.001). The pattern of results was confirmed in multivariate analysis, except that the differences between Caucasians and African Americans was no longer significant. In conclusion, in an obesity clinic population, elevated aminotransferase levels and hypertriglyceridemia were most common in Hispanics and least common in African Americans.


MYL1501D Insulin Glargine: A Review in Diabetes Mellitus.
Subcutaneous MYL1501D insulin glargine 100 U/mL (hereafter referred to as MYL1501D insulin glargine) [Semglee®] is a long-acting human insulin analogue approved as a biosimilar of insulin glargine 100 U/mL (hereafter referred to as reference insulin glargine 100 U/mL) [Lantus®] in various countries, including those of the EU for the treatment of diabetes mellitus in patients aged ≥ 2 years, as well as Japan for diabetes where insulin therapy is indicated. MYL1501D insulin glargine has similar physicochemical characteristics and biological properties to those of EU- and US-sourced reference insulin glargine 100 U/mL, with the bioequivalence of pharmacodynamic and pharmacokinetic parameters between these agents shown in adults with type 1 diabetes. Once-daily MYL1501D insulin glargine demonstrated noninferior glycaemic efficacy to that of once-daily reference insulin glargine 100 U/mL in adults with type 1 or 2 diabetes, with its glycated haemoglobin-lowering benefits maintained over the longer-term (52 weeks) and unaffected by previous insulin exposure. Switching between MYL1501D insulin glargine and reference insulin glargine 100 U/mL did not appear to impact glycaemic efficacy in adults with type 1 diabetes. MYL1501D insulin glargine was well tolerated, demonstrating a safety and immunogenicity profile similar to that of reference insulin glargine 100 U/mL in patients with type 1 and 2 diabetes, and in those with type 1 diabetes switching between the two agents. As expected, hypoglycaemia was the most frequently reported treatment-emergent adverse event. Thus, MYL1501D insulin glargine provides an effective biosimilar alternative for patients requiring insulin glargine therapy.


The Effect of Previsit Education in Breast Cancer Patients: A Study of a Shared-decision-making Tool.
To enhance shared decision-making for patients with breast cancer, we developed an evidence-based educational breast cancer video (BCV) providing an overview of breast cancer biology, prognostic indicators, and surgical treatment options while introducing health care choice. By providing patients access to a BCV with information necessary to make informed surgical decisions before seeing a surgeon, we aimed to increase patient participation in the decision-making process, while decreasing distress. Patients with a new diagnosis of breast cancer were provided a link to the BCV. Group 1 participated in online pre- and postvideo questionnaires, with the BCV embedded in between. The questionnaires evaluated self-reported baseline knowledge of breast cancer and perceived distress related to the diagnosis. Changes in self-reported responses were analyzed using the Wilcoxon matched pairs test. Group 2 received a survey collecting demographics, decision-making information, and perceptions of the BCV at the time of clinic visit before meeting the surgeon. Group 1 included 69 subjects with 62 per cent reporting improved knowledge and 30 per cent reporting reduced distress in regard to their breast cancer diagnosis. Group 2 included 87 subjects; 94 to 98 per cent felt the BCV provided information and stimulated thoughts and questions to assist in breast cancer treatment decision-making. The BCV was positively received by participants and feasible to implement into clinical practice. Evidence-based media tools improve knowledge and reduce distress in patients with a new diagnosis of breast cancer as well as contributing to the shared decision-making process.


Fast generation of virtual X-ray images for reconstruction of 3D anatomy.
We propose a novel GPU-based approach to render virtual X-ray projections of deformable tetrahedral meshes. These meshes represent the shape and the internal density distribution of a particular anatomical structure and are derived from statistical shape and intensity models (SSIMs). We apply our method to improve the geometric reconstruction of 3D anatomy (e.g. pelvic bone) from 2D X-ray images. For that purpose, shape and density of a tetrahedral mesh are varied and virtual X-ray projections are generated within an optimization process until the similarity between the computed virtual X-ray and the respective anatomy depicted in a given clinical X-ray is maximized. The OpenGL implementation presented in this work deforms and projects tetrahedral meshes of high resolution (200.000+ tetrahedra) at interactive rates. It generates virtual X-rays that accurately depict the density distribution of an anatomy of interest. Compared to existing methods that accumulate X-ray attenuation in deformable meshes, our novel approach significantly boosts the deformation/projection performance. The proposed projection algorithm scales better with respect to mesh resolution and complexity of the density distribution, and the combined deformation and projection on the GPU scales better with respect to the number of deformation parameters. The gain in performance allows for a larger number of cycles in the optimization process. Consequently, it reduces the risk of being stuck in a local optimum. We believe that our approach will improve treatments in orthopedics, where 3D anatomical information is essential.


Thirty-day postoperative complications and mortality following total knee arthroplasty: incidence and risk factors among a national sample of 15,321 patients.
The purpose of this investigation was to determine the incidence rates of, and identify risk factors for, thirty-day postoperative mortality and complications among more than 15,000 patients who underwent a primary unilateral total knee arthroplasty as documented in the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP). The NSQIP database was queried to identify patients who had undergone primary unilateral total knee arthroplasty between 2006 and 2010. Patient demographics, medical history, and surgical characteristics were recorded, as were thirty-day postoperative complications, mortality, and length of hospital stay. Complications were divided into categories, which included major systemic complications (complications requiring complex medical intervention) and major local complications (including deep wound infection and peripheral nerve injury). Univariate testing and multivariate logistic regression analysis were used to identify significant independent predictors of the outcome measures. A total of 15,321 individuals underwent primary unilateral total knee arthroplasty. The mean age (and standard deviation) of the patients was 67.3 ± 10.2 years. Obesity (a body mass index [BMI] of ≥30 kg/m²) was documented in 61.2% of cases, 18.2% of patients had diabetes, and 50% were graded as Class 3 or higher on the basis of the American Society of Anesthesiologists (ASA) classification system. The thirty-day mortality rate was 0.18%, and 5.6% of the patients experienced complications. Patient age (odds ratio [OR] = 1.12; 95% confidence interval [CI] = 1.06 to 1.17) and diabetes (OR = 2.99; 95% CI = 1.35 to 6.62) were independent predictors of mortality. A BMI of ≥40 kg/m² was an independent predictor of postoperative complications (OR = 1.47; 95% CI = 1.09 to 1.98). Patient age of eighty years or older, an ASA classification of ≥3, and an operative time of >135 minutes influenced the development of any postoperative complication as well as major and minor systemic complications. Cardiac disease (OR = 4.32; 95% CI = 1.01 to 18.45) and a BMI of ≥40 kg/m² (OR = 2.01; 95% CI = 1.02 to 3.97) were associated with minor local complications. Patient age and diabetes increased the risk of mortality after primary total unilateral knee arthroplasty. Predictive factors impacting the development of postoperative complications included an ASA classification of ≥3, increased operative time, increased age, and greater body mass.


Association between serum interleukin (IL)-12 level and severity of non-alcoholic fatty liver disease (NAFLD).
What is new? Serum IL-12 level is associated with NAFLD severity. Elevation in serum IL-12 level is in line with more severe NAFLD based on BARD score and NAFLD fibrosis score. Positive correlation is observed between serum IL-12 level and BARD score.Introduction. Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. Lipid accumulation in the liver triggers inflammation and leads to NAFLD. Prolonged inflammation will worsen the disease progression. Pro-inflammatory cytokines, including interleukin (IL)-12, plays a role in the inflammatory process. This study aimed to determine the association between IL-12 and NAFLD severity.Methods. A cross-sectional study was conducted between January and July 2019 in Haji Adam Malik Hospital Medan, Indonesia. Subjects were patients aged 18 years or older diagnosed with NAFLD based on ultrasound. Exclusion criteria were excessive alcohol consumption, other primary liver diseases, malignancies, and cardio-metabolic disturbances. Serum IL-12 level was determined using an enzyme-linked immunosorbent assay method. The severity of NAFLD was assessed using the BARD score and NAFLD fibrosis score.Results. A total of 100 subjects were enrolled with male predominant. The mean age of subjects was 54.97 ± 8.85 years, and the most frequent comorbidity was obesity. Most subjects had mild to moderate disease progression. Serum IL-12 level was higher in more severe NAFLD based on ultrasound grading (P < 0.001), BARD score (P = 0.003), and NAFLD fibrosis score (P = 0.005). A positive correlation was observed between serum IL-12 level and BARD score (P < 0.001) with sufficient accuracy (AUC = 0.691, P = 0.014).Conclusion. Serum IL-12 level was associated with the severity of NAFLD. Higher serum IL-12 level was observed in more severe NAFLD progression.


Silica hybrid for corneal replacement: optical, biomechanical, and ex vivo biocompatibility studies.
To investigate compositions of silica-collagen hybrid materials as potential artificial corneal substitutes, how these components affect the optical and biomechanical properties of the hybrids, and their biocompatibility in an organ culture model. Hybrid materials were created from different proportions of collagen and silica precursors and manufactured to specific dimensions. The microstructure of the materials was determined by electron microscopy and mechanical strength was measured by using suture pullout tests. The refractive index and transmittance were measured by using an Abbe refractometer and a spectrophotometer. Materials were implanted into rabbit corneas to determine their epithelialization in organ culture. Scanning electron microscopy demonstrated that the hybrid material consisted of silica-encapsulating collagen fibrils. The refractive index ranged from 1.332 to 1.403 depending upon the composition and manufacturing characteristics. The rupture strength of a 3:1 (silica:collagen ratio by weight) rehydrated xerogel was 0.161 ± 0.073 N/mm (n = 12), while the hydrogels and 9:1 xerogel were too fragile for suturing. Re-epithelialization of 5- to 6-mm-wide rabbit corneal epithelial defects was complete in 5.5 ± 2.4 days (n = 6), with evidence of epithelial stratification. Silica-collagen hybrid materials can be manufactured to specific dimensions to serve as a possible artificial corneal substitute. In preliminary studies, the materials had favorable optical, biomechanical, and biocompatibility properties necessary for replacing the corneal stroma.


On gray dancing: Constructions of age-normality through choreography and temporal codes.
Against the background of population aging, older peoples dance has attracted attention in research and its health promoting effects and social meanings have been brought to the fore. In this article we focus on the context and power dimensions of dance with an emphasis on the organizing of dance among older adults in terms of social discourses and age relationships. On the basis of qualitative interviews with 33 older dancers and 11 dance providers in Sweden, the study illustrates how dance is organized through social discourses on healthism and on the increasing group of older people as a powerful consumer group. The study highlights that older people and their social dance contexts are marked and subordinated in relation to younger age groups through non-verbal practices such as choreography and temporal codes. In short, dancing among older adults is not only a common health promoting and social activity, but also an arena in which age and age normality are negotiated and constructed.


Laparoscopic reduction of intussusception: an evolving therapeutic option.
Intussusception (IS) is a common cause of bowel obstruction in the pediatric population. Traditionally, unsuccessful hydrostatic reduction has been followed by laparotomy. With the advent of minimally invasive surgery, centers have adopted laparoscopic reduction as a surgical option. We reviewed our experience with IS and investigated whether there were any advantages to performing laparoscopy over conventional laparotomy in unsuccessful air enema reduction (AE). All the records of patients admitted from January 2001 to August 2004 with a diagnosis of IS (diagnosis code 560.0) were reviewed. Parameters investigated included age, sex, weight, radiological intervention, operative procedure, length of stay (LOS), and days to oral intake (p.o.). Statistical analysis was performed with the 2-tailed t test to compare outcomes and Fisher's exact test to assess differences in nominal frequencies. Seventeen males and 9 females diagnosed with IS were identified. The mean age was 2.5 years (range, 1 month to 14 years), and the average weight was 5.65 kg (range, 4.65 to 95). Twenty-three of the 26 patients (88.5%) underwent AE reduction, with success in 13 (57%). One recurred after initial successful AE, 9 failed multiple attempts at AE, and 2 attempted reductions were complicated by perforations. Fifteen patients underwent surgical reduction for unsuccessful AE or to address a pathological lead point. The success rate of laparoscopic reduction was 85%. The average time to resumption of p.o. intake for patients with successful AE was 0.5 days, and after laparoscopic reduction, the average time to p.o. intake was 1.5 days, while it was 4 days after laparotomy (P=0.05). After laparoscopic reduction, the average LOS was 6 days, but LOS was 7 days after laparotomy (P=0.66). Many children who present with IS can be treated by AE. In patients who fail AE, laparoscopy offers a safe, effective alternative to laparotomy.


Visual Sonority Modulates Infants' Attraction to Sign Language.
The infant brain may be predisposed to identify perceptually salient cues that are common to both signed and spoken languages. Recent theory based on spoken languages has advanced sonority as one of these potential language acquisition cues. Using a preferential looking paradigm with an infrared eye tracker, we explored visual attention of hearing 6- and 12-month-olds with no sign language experience as they watched fingerspelling stimuli that either conformed to high sonority (well-formed) or low sonority (ill-formed) values, which are relevant to syllabic structure in signed language. Younger babies showed highly significant looking preferences for well-formed, high sonority fingerspelling, while older babies showed no preference for either fingerspelling variant, despite showing a strong preference in a control condition. The present findings suggest babies possess a sensitivity to specific sonority-based contrastive cues at the core of human language structure that is subject to perceptual narrowing, irrespective of language modality (visual or auditory), shedding new light on universals of early language learning.


Brief report: Utility of a short screening scale for DSM-IV PTSD in primary care.
To evaluate Breslau's 7-item screen for posttraumatic stress disorder (PTSD) for use in primary care. One hundred and thirty-four patients were recruited from primary care clinics at a large medical center. Participants completed the self-administered 7-item PTSD screen. Later, psychologists blinded to the results of the screen-interviewed patients using the Clinician Administered PTSD Scale (CAPS). Sensitivity, specificity, and likelihood ratios (LR) were calculated using the CAPS as the criterion for PTSD. The screen appears to have test-retest reliability (r=.84), and LRs range from 0.04 to 13.4. Screening for PTSD in primary care is time efficient and has the potential to increase the detection of previously unrecognized PTSD.


Allyl isothiocyanate-rich mustard seed powder inhibits bladder cancer growth and muscle invasion.
Allyl isothiocyanate (AITC), which occurs in many common cruciferous vegetables, was recently shown to be selectively delivered to bladder cancer tissues through urinary excretion and to inhibit bladder cancer development in rats. The present investigation was designed to test the hypothesis that AITC-containing cruciferous vegetables also inhibit bladder cancer development. We focused on an AITC-rich mustard seed powder (MSP-1). AITC was stably stored as its glucosinolate precursor (sinigrin) in MSP-1. Upon addition of water, however, sinigrin was readily hydrolyzed by the accompanying endogenous myrosinase. This myrosinase was also required for full conversion of sinigrin to AITC in vivo, but the matrix of MSP-1 had no effect on AITC bioavailability. Sinigrin itself was not bioactive, whereas hydrated MSP-1 caused apoptosis and G(2)/M phase arrest in bladder cancer cell lines in vitro. Comparison between hydrated MSP-1 and pure sinigrin with added myrosinase suggested that the anticancer effect of MSP-1 was derived principally, if not entirely, from the AITC generated from sinigrin. In an orthotopic rat bladder cancer model, oral MSP-1 at 71.5 mg/kg (sinigrin dose of 9 μmol/kg) inhibited bladder cancer growth by 34.5% (P < 0.05) and blocked muscle invasion by 100%. Moreover, the anticancer activity was associated with significant modulation of key cancer therapeutic targets, including vascular endothelial growth factor, cyclin B1 and caspase 3. On an equimolar basis, the anticancer activity of AITC delivered as MSP-1 appears to be more robust than that of pure AITC. MSP-1 is thus an attractive delivery vehicle for AITC and it strongly inhibits bladder cancer development and progression.


Niacin depletion in Parkinsonian patients treated with L-dopa, benserazide and carbidopa.
1. Benserazide and carbidopa, decarboxylase inhibitors used in the treatment of Parkinson's disease, have been shown to inhibit the enzyme kynurenine hydrolase in rat and mouse liver. This results in reduced synthesis of nicotinamide coenzymes from tryptophan, and hence an increased reliance on dietary niacin. 2. Pellagra might be expected as a result of this inhibition of endogenous synthesis of nicotinamide nucleotides, but has not been reported in patients treated with either drug. 3. The urinary excretion of N1-methyl-nicotinamide, a product of nicotinamide nucleotide metabolism, is considerably reduced in patients treated with dopa alone or in combination with an inhibitor of peripheral dopa decarboxylase, to as low as 40% of the control value. This means that many of these patients could be classified as 'at risk' of niacin deficiency, even if not frankly deficient. 4. Patients treated with dopa plus a decarboxylase inhibitor, but not those treated with dopa alone, also show a reduced excretion of xanthurenic acid, and an increased excretion of kynurenine, as would be expected after inhibition of the kynurenine pathway, and possibly indicative of marginal vitamin B6 deficiency.


[The development of transaminase activity (SGOT and SGPT), lactate dehydrogenase and alkaline phosphatase in the blood plasma of calves up to the age of 4 months].
A trial was performed in 204 healthy calves (heifers) of the Bohemian Spotted breed in the post-natal period from birth to the age of four months. The activities of the following enzymes in blood plasma were determined: L-aspartate: 2-oxoglutarate: aminotransferase, EC.22.214.171.124. (GOT), L-alanine: oxoglutarate: aminotransferase, EC.220.127.116.11. (GPT), L-lactate: NAD oxidoreductase, EC.188.8.131.52 (LDH), and orthophosphoric acid monoester phosphohydrolase, EC.126.96.36.199. (alkaline phosphatase). The calves were divided into age categories according to the date of birth with an interval of one week. GOT activity in blood plasma increased significantly until the age of eight weeks (from the original value of 1.1708 +/- 0.2598 micronmol ml-1 to 1.8150 +/- 0.6362 micronmol ml-1, with the maximum of 2.0317 +/- 0.7777 micronmol ml-1 of plasma in the sixth week). In the subsequent period the GOT curve has not a characteristic course. While the activity of GOT increased in the first weeks after birth, the activity of GPT showed a significant drop (from the original level of 0.9000 +/- 0.3364 micronmol ml-1 to the minimum of 0.3675 +/- 0.1901 micronmol ml-1 of plasma in the seventh week); from the 10th week on the values rise so that at the end of the period of study they reach almost the same levels as in calves in the first postnatal week. The activity of LDH in blood plasma remains at almost the same level in the first five weeks after birth (between 43.4025 +/- 8.4893 micronmol ml-1 and 46.3792 +/- 14.8952 micronmol ml-1 of plasma); it was at a statistically significantly higher level only in a short period between the 7th and 10th week after birth. The highest values of alkaline phosphatase in blood plasma were recorded at the age of two or three weeks (maximum in the second week 23.9833 +/- 9.0945 micronmol ml-1 of plasma); from the fourth week on, the values of alkaline phosphatase are significantly lower until the end of the test period, ranging betweek 5.3133 +/- 1.6017 micronmol ml-1 and 7.5425 +/- 2.2437 micronmol ml-1 of plasma. Changes conditioned by postnatal development were observed in the development of all the enzymatic activities under study, the greatest changes being observed in alkaline phosphatase.


Reverse phase protein array profiling reveals distinct proteomic signatures associated with chronic myeloid leukemia progression and with chronic phase in the CD34-positive compartment.
Chronic myeloid leukemia (CML) is a clonal stem cell malignancy whose pathogenesis is driven by constitutive activation of the breakpoint cluster region-v-abl Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1) kinase. Although BCR-ABL1 activation is present in all patients with CML, patients can present in 3 different phases characterized by an increasingly worse prognosis and diminished responsiveness to tyrosine kinase inhibitors: chronic phase, accelerated phase, or blastic phase. The biologic basis for progression from chronic phase to blastic phase and for regulating the homeostasis of tyrosine kinase inhibitor-resistant CML stem cells is not entirely understood. To shed some light into these aspects of CML biology, the authors used reverse phase protein arrays probed with 112 individual monoclonal antibodies to compare protein expression patterns in 40 samples of leukemia-enriched fractions from patients with CML (25 in chronic phase, 5 in accelerated phase, and 10 in phase). An analysis of variance (significance cutoff, P < .01) unveiled a set of proteins that were overexpressed in blastic phase, including heat-shock protein 90 (hsp90); retinoblastoma (Rb); apoptosis-inducing factor (AIF); serine/threonine-protein phosphatase 2A (PP2A); B-cell leukemia 2 (Bcl-2); X-linked inhibitor of apoptosis protein (Xiap); human homolog of Drosophila Mad (mothers against decapentaplegic) and related Caenorhabditis elegans gene Sma, family member 1 (Smad1); single-stranded DNA binding protein 2 alpha (SSBP2α); poly(adenosine diphosphate-ribose) polymerase (PARP); GRB2-associated binding protein 2 (Gab2); and tripartite motif containing 24 (Trim24). It is noteworthy that several of these proteins also were overexpressed in the CD34-positive compartment, which putatively contains the CML stem cell population. The results from this study indicated that reverse phase protein array analysis can unveil differentially expressed proteins in advanced phase CML that can be exploited therapeutically with targeted approaches.


Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2.
Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A-TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A-TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation.


Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide.
The class 1 outer-membrane protein of Neisseria meningitidis is the target for subtype-specific, bactericidal monoclonal antibodies (mAbs). The epitopes recognized by these antibodies have been mapped previously to linear peptides corresponding to the sequences thought to be exposed at the apices of surface-exposed loops of the protein. In this work several synthetic peptides containing the subtype Pl.16b epitope have been synthetized with the aim of inducing a polyclonal immune response resembling the reactivity of the mAbs. Initially, peptides of 9 and 15 amino acid residues were synthesized and used for immunization after coupling to a carrier protein. The reactivity of the resulting antisera, with synthetic linear decapeptides, resembled that seen in previous epitope mapping experiments with the protective mAbs. However, despite the induction of antibodies having the desired specificity, the antisera reacted poorly with the native protein in outer membranes, and were non-bactericidal. A 36mer peptide, consisting of the entire surface-exposed loop 4 of the class 1 protein was then synthesized and used for immunization as (i) free peptide, (ii) peptide coupled to carrier and (iii) peptide subjected to cyclization, in an attempt to restrict it to conformations that might more closely resemble the native loop structure. In contrast to antisera raised against linear peptides, antibodies raised by immunization with the 36mer cyclic peptide, did not react with linear peptides recognized by the mAbs, but instead appeared to recognize conformational determinants. This antiserum promoted complement-mediated bactericidal killing of the homologous meningococcal strain, demonstrating the potential of synthetic peptide immunogens for inducing a protective immune response against group B meningococci.


Induction of collagenolytic and proteolytic activities in rat and human fibroblasts by anti-inflammatory drugs.
Confluent monolayers of either mouse or diploid human fibroblasts contained no measurable amounts of either collagenolytic (pH 5.5) or proteolytic (pH 7.5) activities. Within a few hours after exposure of these cells to antiinflammatory drugs, significant amounts of these enzymatic activities were demonstrable extracellularly. These activities were profoundly decreased in cultures simultaneously treated with the anti-inflammatory drugs and cycloheximide or actinomycin D.


Clinical, enzymatic and molecular characterization of nine new patients with malonyl-coenzyme A decarboxylase deficiency.
We report nine new patients with malonic aciduria associated with enzyme-confirmed malonyl-CoA decarboxylase (MCD) deficiency in eight. Clinical details were available on eight, and molecular genetic characterization was obtained for nine. As for 15 previously described patients, cardinal clinical manifestations included developmental delay and cardiomyopathy; metabolic perturbations (e.g. acidosis) and seizures, however, were infrequent or not observed in our patients. For all, detection of elevated malonic acid in urine (+/- increased C3DC acylcarnitine by analysis employing tandem mass spectrometry) led to pursuit of enzyme studies. MCD activities (nmol/h PER mg protein) revealed: control (n = 22), 16.2 +/- 1.8 (SEM; range 5.7-46.2); patients (n = 8, assayed in duplicate), 1.7 +/- 0.3 (10% of parallel control; range 0.6-2.8). Molecular characterization by DNA sequence analysis and multiplex ligation-dependent probe amplification revealed nine novel mutations (c.796C>T; p.Gln266X, c.481delC; p.Leu161CysfsX18, c.1367A>C; p.Tyr456Ser, c.1319G>T; p.Ser440Ile, c.1430C>T; p.Ser477Phe, c.899G>T; p.Gly300Val, c.799-1683_949-1293del3128, and two other large genomic deletions comprising exons 1 or the complete gene) and two known mutations in the MLYCD gene. Our findings increase the number of enzyme-confirmed MCD-deficient patients by >50%, and expand our understanding of the phenotypic and molecular heterogeneity of this rare disorder.


A hook effect-free immunochromatographic assay (HEF-ICA) for measuring the C-reactive protein concentration in one drop of human serum.
The immunochromatographic (ICA) assay is a highly promising platform for rapid and simple detection of C-reactive protein (CRP) which is an indicator of the different phases of various diseases, as well as of inflammation and infection. However, the hook effect in the ICA assay limits the quantification of CRP levels at high CRP concentrations. Methods: In this study, we developed a hook effect-free immunochromatographic assay (HEF-ICA) to detect CRP over a wide concentration range. The hook effect results from the simultaneous reaction of an excess target antigens with both immobilized and labeled antibodies respectively. To reduce the potential occurrence of this simultaneous reaction, we separated the migration of the target antigen and gold nanoparticle (AuNP)-labeled antibodies on a nitrocellulose membrane and analyzed the time profiles by modifying the ICA structure. Results: The signal intensity of HEF-ICA was saturated at high CRP concentrations, without decreasing. The titration curve of HEF-ICA was adjusted with the Hill equation, and HEF-ICA was performed with the following parameters: limit of detection, 43 ng mL-1; dynamic range, 119 ng mL-1 to 100 µg mL-1. The accuracy of the newly developed assay was evaluated using 33 clinical samples via comparison with a clinical chemistry analyzer. Conclusion: HEF-ICA enabled the measurement of a wide range of CRP concentrations without the hook effect, and was suitable for point-of-care testing with fingertip blood sampling, as only a minute sample volume (2.5 µL) was required.


Long-term effect of smartphone-delivered Interval Walking Training on physical activity in patients with type 2 diabetes: protocol for a parallel group single-blinded randomised controlled trial.
Physical activity is a cornerstone in type 2 diabetes (T2D) rehabilitation. Effective long-term and low-cost strategies to keep these patients' physically active are needed. However, maintaining physical activity behaviour is difficult once formalised interventions end. Structured exercise training supported by mobile technology and remote feedback is potentially an effective strategy. The objective of the trial is to investigate whether mobile health support using the InterWalk application for smartphones is effective in increasing physical activity levels in persons with T2D over time compared with standard care. We investigate whether Interval Walking Training using the InterWalk application is superior to Danish municipality-based rehabilitation in increasing moderate-and-vigorous physical activity levels in patients with T2D across 52 weeks. Secondary, we hypothesise that a motivational programme added from end of intervention to 52 weeks further increases level of physical activity in everyday life in patients with T2D. The trial is a parallel-group, open-labelled, randomised controlled trial with long-term follow-up at 52 week including patients with T2D. The primary outcome is change in moderate-and-vigorous physical activity. The key secondary outcome includes motivation for physical activity behaviour change. Other secondary outcomes are VO2-peak, strength in the lower extremities. Exclusion criterion is medical contraindication to exercise. We include up to 246 patients and randomly allocate them into a control (standard group) or an experimental group (8-12 weeks of IWT supported by the smartphone-based InterWalk application) in a 1:2 fashion. After intervention, the experimental group is randomly allocated into two follow-up conditions with unsupervised IWT with or without motivational support until 52-week follow-up. The intention-to-treat principle is applied. The local regional Research Ethics Committee in Denmark (H-1-2014-074) and the Danish Data Protection Agency (j.nr. 2014-54-0897) have approved the trial. Positive, negative or inconclusive results will be disseminated in scientific journals and conferences. NCT02341690.


1-(2'-Anilinyl)prop-2-yn-1-ol rearrangement for oxindole synthesis.
A synthetic method that relies on NIS (N-iodosuccinimide)-mediated cycloisomerization reactions of 1-(2'-anilinyl)prop-2-yn-1-ols to gem-3-(diiodomethyl)indolin-2-ones and 2-(iodomethylene)indolin-3-ones has been developed. The reactions were shown to be chemoselective, with secondary and tertiary alcoholic substrates exclusively giving the 3- and 2-oxindole products, respectively. In the case of the latter, the transformation features an unprecedented double 1,2-OH and 1,2-alkyl migration relay. Density functional theory (DFT) calculations based on proposed iodoaminocyclization species provide insight into this unique divergence in product selectivity.


The Lived Experiences of Mothers Who Are Parenting Children With Leukemia.
Mothers parenting children with leukemia face innumerable challenges in meeting their own personal needs, as well as the needs of their ill child. To explore the lived experiences of Omani mothers who are parenting children with leukemia, using interpretative phenomenological analysis design. Data were collected from 10 mothers who were parenting children with leukemia who shared their experiences through a face-to-face recorded interview. Data were analyzed using the interpretative phenomenological analysis approach methods. Three main themes emerged from the mothers' experiences: (1) unexpected shocking diagnosis; (2) life is not the same anymore; and (3) viable support systems. Mothers found their child's diagnosis to be shocking and terrifying. The diagnosis and treatment cast a shadow on the mothers' lives. Mothers abandoned their work and left their healthy children to their spouses. Mothers relied on other mothers also parenting children with cancer and on the support of the extended family and health team members to cope and prayed to God to help them overcome the calamity. The mothers perceived their life as changing immediately upon hearing their child's cancer diagnosis as they experienced a plethora of emotions. They sought support at various points during their child's illness to cope and overcome the challenges during the cancer treatment journey. Pediatric oncology nurses can support mothers during their transition to caring for a child with leukemia and can provide the needed support to reduce maternal distress and increase effective coping with the challenges.