Babesia bigemina: sporozoite isolation from Boophilus microplus nymphs and initial immunomolecular characterization.
It has been hypothesized that babesial sporozoites express specific antigens that induce protective immunologic responses in cattle. However, they remain uncharacterized, partly for lack of research on the sporozoite stage of Babesia spp. This field suffers from complete knowledge of parasite development in the tick salivary gland; limited amounts of sporozoites from ticks, and a lack of protocols for induction and purification of sporozoites. In this work, Boophilus microplus larvae infected with B. bigemina were fed on susceptible cattle. Nymphs were collected and macerates were separated by a Percoll density gradient. Microscopic analysis of Giemsa-stained smears showed a larger number of sporozoites from nymphs fed for 9 days. Percoll-purified sporozoites were observed in large numbers in groups or individually and free of tick cells. RT-PCR analysis of total RNA extracted from purified sporozoites indicated transcription of the rhoptry associate protein 1 (rap-1) genes: rap-1a, rap-b, rap-1c, as well as the heat shock protein 20 (hsp-20) gene. Purified sporozoites were cultured in vitro analyzed for RAP-1a expression using an immunocytochemistry assay. Erythrocyte-attached sporozoites reacted with a specific RAP-1a monoclonal antibody. This is the first report of Babesia bigemina sporozoite antigens. Moreover, purified sporozoites will allow the characterization of stage-specific antigens involved in immunologic protection.


Probing the ArcA regulon under aerobic/ROS conditions in Salmonella enterica serovar Typhimurium.
Hydrogen peroxide (H₂O₂) is a reactive oxygen species (ROS), which is part of the oxidative burst encountered upon internalization of Salmonella enterica serovar Typhimurium (S. Typhimurium) by phagocytic cells. It has previously been established that, the ArcAB two-component system plays a critical role in ROS resistance, but the genes regulated by the system remained undetermined to date. We therefore investigated the ArcA regulon in aerobically growing S. Typhimurium before and after exposure to H₂O₂ by querying gene expression and other physiological changes in wild type and ΔarcA strains. In the ΔarcA strain, expression of 292 genes showed direct or indirect regulation by ArcA in response to H₂O₂, of which 141were also regulated in aerobiosis, but in the opposite direction. Gene set enrichment analysis (GSEA) of the expression data from WT and ΔarcA strains, revealed that, in response to H₂O₂ challenge in aerobically grown cells, ArcA down regulated multiple PEP-PTS and ABC transporters, while up regulating genes involved in glutathione and glycerolipid metabolism and nucleotide transport. Further biochemical analysis guided by GSEA results showed that deletion of arcA during aerobic growth lead to increased reactive oxygen species (ROS) production which was concomitant with an increased NADH/NAD+ ratio. In absence of ArcA under aerobic conditions, H₂O₂ exposure resulted in lower levels of glutathione reductase activity, leading to a decreased GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio. The ArcA regulon was defined in 2 conditions, aerobic growth and the combination of peroxide treatment and aerobic growth in S. Typhimurium. ArcA coordinates a response that involves multiple aspects of the carbon flux through central metabolism, which ultimately modulates the reducing potential of the cell.


The use of a Doppler ultrasound probe during vascular dissection in laparoscopic renal surgery.
There have been many reports regarding the use of intraoperative ultrasound to identify vascular structures. However, few have examined the application of Doppler technology for the identification and dissection of hilar vessels during laparoscopic renal surgery (LRS). We describe the design and utility of what is, to our knowledge, the only available disposable laparoscopic Doppler probe (LDP). A disposable 18-inch, 8-MHz LDP (Vascular Technology, Inc., Nashua, NH) was inserted through a 5-mm laparoscopic port during LRS and moved systematically in a cephalad direction along the medial aspect of the kidney in an attempt to obtain an audible signal from the vessels of the renal hilum. Various endpoints such as ease of probe use, time to identification of vessels, and detection of aberrant vessels were recorded. A total of 20 patients underwent LRS by a single surgeon (C.P.S.). The LDP was shown to be very easy to use in the identification of the renal hilum. The mean time for the identification of all renal vessels by LDP survey was 34.9 seconds. The mean time to surgical isolation of all hilar vessels was 44.6 minutes. There were no technical difficulties or complications as a result of Doppler probe use. The use of the LDP provides a fast, efficient, and simple means of identifying the renal hilum during LRS. This technology may have added benefit and utility in patients with anomalous vessels, significant adhesions, or during laparoscopic surgery by less experienced surgeons.


Prm1 prevents contact-dependent lysis of yeast mating pairs.
Membrane fusion requires localized destabilization of two phospholipid bilayers, but unrestrained membrane destabilization could result in lysis. prm1 mutant yeast cells have a defect at the plasma membrane fusion stage of mating that typically results in the accumulation of prezygotes that have fingers of membrane-bound cytoplasm projecting from one cell of each pair into its mating partner in the direction of the osmotic gradient between the cells. However, some prm1 mating pairs fuse successfully whereas the two cells in other prm1 mating pairs simultaneously lyse. Lysis only occurs if both mating partners are prm1 mutants. Osmotic stabilization does not protect prm1 mating pairs from lysis, indicating that lysis is not caused by a cell wall defect. prm1 mating pairs without functional mitochondria still lyse, ruling out programmed cell death. No excess lysis was found after pheromone treatment of haploid prm1 cells, and lysis did not occur in mating pairs when prm1 was combined with the fus1 and fus2 mutations to block cell wall remodeling. Furthermore, short (<1 microm) cytoplasmic microfingers indicating the completion of cell wall remodeling appeared immediately before lysis. In combination, these results demonstrate that plasma membrane contact is a prerequisite for lysis. Cytoplasmic microfingers are unlikely to cause lysis since most prm1 mating pairs with microfingers do not lyse, and microfingers were also detected before fusion in some wild-type mating pairs. The lysis of prm1 mutant mating pairs suggests that the Prm1 protein stabilizes the membrane fusion event of yeast mating.


Rational Design and Development of Anisotropic and Mechanically Strong Gelatin-Based Stress Relaxing Hydrogels for Osteogenic/Chondrogenic Differentiation.
Rational design and development of tailorable simple synthesis process remains a centerpiece of investigational efforts toward engineering advanced hydrogels. In this study, a green and scalable synthesis approach is developed to formulate a set of gelatin-based macroporous hybrid hydrogels. This approach consists of four sequential steps starting from liquid-phase pre-crosslinking/grafting, unidirectional freezing, freeze-drying, and finally post-curing process. The chemical crosslinking mainly involves between epoxy groups of functionalized polyethylene glycol and functional groups of gelatin both in liquid and solid state. Importantly, this approach allows to accommodate different polymers, chitosan or hydroxyethyl cellulose, under identical benign condition. Structural and mechanical anisotropy can be tuned by the selection of polymer constituents. Overall, all hydrogels show suitable structural stability, good swellability, high porosity and pore interconnectivity, and maintenance of mechanical integrity during 3-week-long hydrolytic degradation. Under compression, hydrogels exhibit robust mechanical properties with nonlinear elasticity and stress-relaxation behavior and show no sign of mechanical failure under repeated compression at 50% deformation. Biological experiment with human bone marrow mesenchymal stromal cells (hMSCs) reveals that hydrogels are biocompatible, and their physicomechanical properties are suitable to support cells growth, and osteogenic/chondrogenic differentiation, demonstrating their potential application for bone and cartilage regenerative medicine toward clinically relevant endpoints.


APOE epsilon4 and cognitive decline in older stroke patients with early cognitive impairment.
Dementia is common post stroke, but the potential role of early cognitive impairment and APOE epsilon4 as risk factors is unclear. Stroke survivors older than 75 years without dementia at 3 months post stroke received a detailed neuropsychological evaluation at 3 and 15 months post stroke, which included the Cambridge Assessment of Mental Disorders in the Elderly (CAMCOG). Early cognitive impairment was diagnosed using the criteria for cognitive impairment/no dementia (vascular CIND). APOE genotype was determined using a standardized method. One hundred thirty-seven older stroke patients without dementia (mean age 80.6 +/- 4.3, mean CAMCOG score 83.5 +/- 10.4, 68 women) participated in the study, of whom 40 met the criteria for CIND. Stroke patients with one or more APOE epsilon4 alleles were significantly more likely to have CIND (14/40 vs 17/97, odds ratio = 2.5, 95% CI 1.1 to 5.8). Over the 1 year of follow-up, CIND patients with one or more APOE epsilon4 alleles had a mean decline on the total CAMCOG of 2.7 points compared with an improvement of >4 points among patients without APOE epsilon4 (T = 2.9 p = 0.006). CIND patients with an APOE epsilon4 allele also experienced greater decline in memory (T = 2.5, p = 0.015). In older stroke patients with early cognitive impairment, the presence of an APOE epsilon4 allele is associated with greater progression of cognitive decline. This has implications for interventions aimed at the secondary prevention of dementia in stroke patients.


Healthcare professionals' perceptions of a digital parental support, Childbirth Journey, constructed as a serious game-An intervention study.
Globally, the digital sources developed and available in antenatal care differ, and infrastructure challenges may impede the further development of such sources. Challenges accompanying digital developments can include the commonly occurring high workload, which affects healthcare professionals' ability to acquire professional knowledge about how to best support parents in using digital sources. Including healthcare professionals in the development process of digital sources may increase the likelihood that such sources will be adopted and employed by these professionals in their future care work. Therefore, the present research explored healthcare professionals' perceptions of the digital support intervention Childbirth Journey, which was constructed as a serious game for expectant parents. Data were collected through semi-structured focus-group interviews with 11 midwives at antenatal, labour and postnatal clinics as well as with child healthcare nurses. Prior to the interviews, all participants were provided the intervention, Childbirth Journey, which is a serious game in a mobile application format consisting of two distinct parts: (1) a story-driven game and (2) a Knowledge Portal. The data were analysed using phenomenographic methods. The perceptions of Childbirth Journey by healthcare professionals, midwives and child healthcare nurses are presented in four descriptive categories: extended professional support, trustworthy contents, diversity or individuality, and both appealing and in need of development. Current study revealed that Childbirth Journey may be utilised as a digital support for parents, allowing healthcare professionals to offer a digital solution as a complementary support to standard, face-to-face meetings with caregivers. However, the research results also revealed that some elements of Childbirth Journey must be improved, thereby representing a main contribution of this study: insights into how to better develop digital tools under the umbrella of health care. Thus, we conclude that in order to create sustainable and safe digital care solutions that function as trustworthy professional supports instead of technical products that risk harming users, the perspectives of both patients and healthcare professionals should be considered in the exploration and development of these solutions.


Effects of long-term, high-altitude hypoxia on the capillarity of the ovine fetal heart.
To determine the effect of chronic hypoxia on myocardial capillarity, we exposed pregnant ewes to an altitude of 3,820 m from day 30 to day 139 of gestation and compared the fetus to low-altitude (approximately 300 m) controls. We hypothesized that capillarity would increase in the hypoxic myocardium to optimize oxygen and metabolite flux to hypoxic tissues. Fetal hearts were fixed by retrograde aortic perfusion and processed for microscopy and stereological evaluation. Fiber cross-sectional area and capillary density were measured and standardized to sarcomere length. Capillary volume density and capillary diameter were measured, capillary-to-fiber ratio and capillary length density were calculated, and the capillary anisotropy coefficient was obtained from a table of known values. Capillary-to-fiber ratio, capillary volume density, and the capillary anisotropy coefficient were not different between hypoxia and control groups. Capillary diameter was significantly larger in the right compared with the left ventricle of hypoxic but not control hearts; fiber cross-sectional area tended to be larger in the right ventricle of both groups, but this was not significant. As a result of larger fiber size, capillary density and capillary length density were significantly smaller in the right ventricle of hypoxic but not control fetal hearts. Contrary to our hypothesis, the ovine fetus does not show morphological adaptation in the myocardium after approximately 109 days of high-altitude hypoxic stress.


Interfacial behaviour of bovine testis hyaluronidase.
The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.


Anti-PD-1 antibody SHR-1210 plus apatinib for metastatic colorectal cancer: a prospective, single-arm, open-label, phase II trial.
In the REGONIVO study, regorafenib combined with nivolumab was effective in the treatment of microsatellite stable (MSS) metastatic colorectal cancer (mCRC), which indicated anti-angiogenic drugs may enhance the efficacy of immune checkpoint inhibitors. Therefore, we designed a single-arm, single-center, open-label, phase II trial to determine the toxicity and efficacy of SHR-1210 (an anti-PD-1 antibody) plus apatinib in MSS mCRC. The sample size was estimated using a Simon Optimum two-stage design. 10 patients were included at the first stage and if one effective patient observed, an additional 19 patients would be added. Patients with MSS mCRC who refractory to second-line treatment or intolerant to standard treatment were given SHR-1210 200 mg every 2 weeks and apatinib 250-375 mg once daily until unacceptable toxicity or disease progression occurred. In our study, the objective response rate was 0% and the disease control rate was 22.2%. The median progression-free survival was 1.83 months (95% confidence interval (CI) 1.80-1.86 months), and the median overall survival was 7.80 months (95% CI 0-17.07). Treatment-related adverse events (AEs) occurred in all patients (100%). The most common treatment-related AEs were hypertension and proteinuria (70% each). Grade 3 AEs were observed in nine patients (9/10, 90%), and the commonest was hypertension (30%). In conclusion, SHR-1210 combined with apatinib has failed to improve the efficacy of treatment of MSS mCRC, and the intolerable toxicity may be the leading cause.


Magnetic Levitation Haptic Augmentation for Virtual Tissue Stiffness Perception.
Haptic-based tissue stiffness perception is essential for palpation training system, which can provide the surgeon haptic cues for improving the diagnostic abilities. However, current haptic devices, such as Geomagic Touch, fail to provide immersive and natural haptic interaction in virtual surgery due to the inherent mechanical friction, inertia, limited workspace and flawed haptic feedback. To tackle this issue, we design a novel magnetic levitation haptic device based on electromagnetic principles to augment the tissue stiffness perception in virtual environment. Users can naturally interact with the virtual tissue by tracking the motion of magnetic stylus using stereoscopic vision so that they can accurately sense the stiffness by the magnetic stylus, which moves in the magnetic field generated by our device. We propose the idea that the effective magnetic field (EMF) is closely related to the coil attitude for the first time. To fully harness the magnetic field and flexibly generate the specific magnetic field for obtaining required haptic perception, we adopt probability clouds to describe the requirement of interactive applications and put forward an algorithm to calculate the best coil attitude. Moreover, we design a control interface circuit and present a self-adaptive fuzzy proportion integration differentiation (PID) algorithm to precisely control the coil current. We evaluate our haptic device via a series of quantitative experiments which show the high consistency of the experimental and simulated magnetic flux density, the high accuracy (0.28 mm) of real-time 3D positioning and tracking of the magnetic stylus, the low power consumption of the adjustable coil configuration, and the tissue stiffness perception accuracy improvement by 2.38 percent with the self-adaptive fuzzy PID algorithm. We conduct a user study with 22 participants, and the results suggest most of the users can clearly and immersively perceive different tissue stiffness and easily detect the tissue abnormality. Experimental results demonstrate that our magnetic levitation haptic device can provide accurate tissue stiffness perception augmentation with natural and immersive haptic interaction.


Perspectives of Singaporean biomedical researchers and research support staff on actual and ideal IRB review functions and characteristics: A quantitative analysis.
Biomedical research is overseen by numerous Institutional Review Boards (IRBs) in Singapore but there has been no research that examines how the research review process is perceived by the local research community nor is there any systematic data on perceptions regarding the review process or other research ethics processes and IRB characteristics. The aim of this study was to ascertain general views regarding the overall perceived value of ethics review processes; to measure perceptions about local IRB functions and characteristics; to identify IRB functions and characteristics viewed as important; and to compare these views with those of other international studies. An online survey was used with the main component being the IRB-Researcher Assessment Tool (IRB-RAT), a validated tool, to evaluate perceptions of ideal and actual IRB functions and characteristics held by Singaporean researchers and research support staff. Data were analysed descriptively first, with mean and SD of each item of IRB-RAT questionnaire reported, excluding the respondents whose answers were unknown or not applicable. The Wilcoxon Sign Rank test was used to compare the ideal and actual ratings of each IRB-RAT item, while the Mann-Whitney U test was used to compare the ratings of each IRB-RAT item between respondents with different characteristics. The Z-test was used to compare the mean ratings of our cohort with the mean ratings reported in the literature. The correlation between our mean ideal scores and those of two international studies also employing the IRB-RAT was examined. Seventy-one respondents completed the survey. This cohort generally held positive views of the impact of the ethics review process on: the quality of research; establishing and maintaining public trust in research; the protection of research participants; and on the scientific validity of research. The most important ideal IRB characteristics were timeliness, upholding participants' rights while also facilitating research, working with investigators to find solutions when there are disagreements, and not allowing biases to affect reviews. For almost all 45 IRB-RAT statements, the rating of the importance of the characteristic was higher than the rating of how much that characteristic was descriptive of IRBs the respondents were familiar with. There was a significant strong correlation between our study's scores on the ideal IRB characteristics and those of the first and largest published study that employed the IRB-RAT, the US National Validation (USNV) sample in Keith-Spiegel et al. [19]. An understanding of the perceptions held by Singaporean researchers and research support staff on the value that the ethics review process adds, their perceptions of actual IRB functions and characteristics as well as what they view as central to high functioning IRBs is the first step to considering the aspects of the review process that might benefit from improvements. This study provides insight into how our cohort compares to others internationally and highlights strengths and areas for improvement of Singapore IRBs as perceived by a small sample of the local research community. Such insights provide a springboard for additional research and may assist in further enhancing good relations so that both are working towards the same end.


Comparison of MR and diskography in detecting radial tears of the anulus: a postmortem study.
Radial tears of the anulus fibrosus, which anatomic studies suggest are a primary event in disk degeneration, can be detected by diskography or MR imaging. We compared the sensitivity of MR and diskography in the detection of anular tears. MR, diskography, and cryomicrotomy anatomic sectioning were performed in eight cadaver lumbar spines. Diskography demonstrated 15 radial tears in 36 intervertebral disks. MR demonstrated 10 of the 15, a sensitivity of 67%. MR (T2-weighted images) in each of the diskographically normal disks showed the high signal intensity characteristic of normal disks. Thirteen of 15 disks from which contrast medium extravasated at diskography had diminished signal intensity in MR images. We conclude that although MR may demonstrate some radial tears of the anulus, and associated changes in the disk, it cannot be used as effectively as diskography to visualize a radial tear.


[Differentiated immunocorrective therapy in nonspecific ulcerative colitis].
A comparative study of the clinico-immunological efficacy of steroid hormones and thymus drugs in 50 patients with unspecific ulcerative colitis of different activity grades revealed that their efficacy was not similar at different phases of the disease and depended on the activity of the inflammatory process. Corticosteroids were more effective in inflammatory-destructive changes in the mucosa of the large intestine while in mild inflammation with prevalence of ulcerative-reparative processes, immunomodulators (thymalin) are preferred.


Microbial distribution and turnover in Antarctic microbial mats highlight the relevance of heterotrophic bacteria in low-nutrient environments.
Maritime Antarctica has shown the highest increase in temperature in the Southern Hemisphere. Under this scenario, biogeochemical cycles may be altered, resulting in rapid environmental change for Antarctic biota. Microbes that drive biogeochemical cycles often form biofilms or microbial mats in continental meltwater environments. Limnetic microbial mats from the Fildes Peninsula were studied using high-throughput 16S rRNA gene sequencing. Mat samples were collected from 15 meltwater stream sites, comprising a natural gradient from ultraoligotrophic glacier flows to meltwater streams exposed to anthropogenic activities. Our analyses show that microbial community structure differences between mats are explained by environmental NH4+, NO3-, DIN, soluble reactive silicon and conductivity. Microbial mats living under ultraoligotrophic meltwater conditions did not exhibit a dominance of cyanobacterial photoautotrophs, as has been documented for other Antarctic limnetic microbial mats. Instead, ultraoligotrophic mat communities were characterized by the presence of microbes recognized as heterotrophs and photoheterotrophs. This suggests that microbial capabilities for recycling organic matter may be a key factor to dwell in ultra-low nutrient conditions. Our analyses show that phylotype level assemblages exhibit coupled distribution patterns in environmental oligotrophic inland waters. The evaluation of these microbes suggests the relevance of reproductive and structural strategies to pioneer these psychrophilic ultraoligotrophic environments.


Model for fitting longitudinal traits subject to threshold response applied to genetic evaluation for heat tolerance.
A semi-parametric non-linear longitudinal hierarchical model is presented. The model assumes that individual variation exists both in the degree of the linear change of performance (slope) beyond a particular threshold of the independent variable scale and in the magnitude of the threshold itself; these individual variations are attributed to genetic and environmental components. During implementation via a Bayesian MCMC approach, threshold levels were sampled using a Metropolis step because their fully conditional posterior distributions do not have a closed form. The model was tested by simulation following designs similar to previous studies on genetics of heat stress. Posterior means of parameters of interest, under all simulation scenarios, were close to their true values with the latter always being included in the uncertain regions, indicating an absence of bias. The proposed models provide flexible tools for studying genotype by environmental interaction as well as for fitting other longitudinal traits subject to abrupt changes in the performance at particular points on the independent variable scale.


Fried syndrome is a distinct X linked mental retardation syndrome mapping to Xp22.
In 1972, Fried described a large Scottish family affected by X linked mental retardation (XLMR), hydrocephalus, and mild facial dysmorphism. The phenotype has considerable similarity to the MASA syndrome, which results from mutations of the L1CAM gene in Xq28, and this family has since been assumed to be an example of this condition. We have reinvestigated the family for linkage to X chromosome markers, and obtained additional clinical information on surviving affected subjects. The phenotype in these patients has evolved into a distinctive syndrome, with severe mental retardation (MR), spastic diplegia, ventricular dilatation, and calcification of the basal ganglia. Linkage to Xq28 markers has been excluded, suggesting that Fried syndrome is not allelic with MASA syndrome. Two point and multipoint linkage analysis indicates that the gene for this condition lies within the interval KAL-DXS989 in Xp22. We propose the designation Fried syndrome to emphasise the disorder's distinctive phenotype.


Streptomyces K15 DD-peptidase-catalysed reactions with ester and amide carbonyl donors.
In water, the purified 26 000-Mr membrane-bound DD-peptidase of Streptomyces K15 hydrolyses the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate (release of D-lactate) and the amide carbonyl donor Ac2-L-Lys-D-Ala-D-Ala (release of D-alanine) with accumulation of acyl- (Ac2-L-Lys-D-alanyl-)enzyme. Whereas hydrolysis of the ester substrate proceeds to completion, hydrolysis of the amide substrate is negligible because of the capacity of the K15 DD-peptidase for utilizing the released D-alanine in a transfer reaction (Ac2-L-Lys-D-Ala-D-Ala + D-Ala----Ac2-L-Lys-D-Ala-D-Ala + D-Ala) that maintains the concentration of the amide substrate at a constant level. In the presence of an amino acceptor X-NH2 (Gly-Gly or Gly-L-Ala) related to the Streptomyces peptidoglycan, both amide and ester carbonyl donors are processed without detectable accumulation of acyl-enzyme. Under proper conditions, the acceptor activity of water and, in the case of the amide substrate, the acceptor activity of the released D-alanine can be totally overcome so that the two substrates are quantitatively converted into transpeptidated product Ac2-L-Lys-D-Ala-NH-X (and hydrolysis is prevented). Experimental evidence suggests that the amino acceptor modifies both the binding of the carbonyl donor to the enzyme and the ensuing rate of enzyme acylation.


CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron 19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells.
An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT ) in the TOP2α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.


The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.
Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p < 0.01 for both). Cartilage explants cultured with MSU crystals had a greater percentage of dead chondrocytes at the articular surface compared to untreated cartilage (p = 0.004). Relative mRNA expression of type II collagen and the cartilage matrix proteins aggrecan and versican was decreased in chondrocytes following culture with MSU crystals (p < 0.05 for all). However, expression of the degradative enzymes ADAMTS4 and ADAMTS5 was increased (p < 0.05 for both). In joints affected by gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.


The diagnosis of attention-deficit/hyperactivity disorder in Australian children: Current paediatric practice and parent perspective.
In a sample of newly diagnosed children with attention-deficit/hyperactivity disorder (ADHD), the aims were to examine (1) paediatrician assessment and management practices; (2) previous assessments and interventions; (3) correspondence between parent-report and paediatrician identification of comorbidities; and (4) parent agreement with diagnosis of ADHD. cross-sectional, multi-site practice audit with questionnaires completed by paediatricians and parents at the point of ADHD diagnosis. private/public paediatric practices in Western Australia and Victoria, Australia. paediatricians: elements of assessment and management were indicated on a study-designed data form. Parents: ADHD symptoms and comorbidities were measured using the Conners 3 ADHD Index and Strengths and Difficulties Questionnaire, respectively. Sleep problems, previous assessments and interventions, and agreement with ADHD diagnosis were measured by questionnaire. Twenty-four paediatricians participated, providing data on 137 patients (77% men, mean age 8.1 years). Parent and teacher questionnaires were used in 88% and 85% of assessments, respectively. Medication was prescribed in 75% of cases. Comorbidities were commonly diagnosed (70%); however, the proportion of patients identified by paediatricians with internalising problems (18%), externalising problems (15%) and sleep problems (4%) was less than by parent report (51%, 66% and 39%). One in seven parents did not agree with the diagnosis of ADHD. Australian paediatric practice in relation to ADHD assessment is generally consistent with best practice guidelines; however, improvements are needed in relation to the routine use of questionnaires and the identification of comorbidities. A proportion of parents do not agree with the diagnosis of ADHD made by their paediatrician.


Strategies for tricuspid re-repair in Ebstein malformation using the cone technique.
Successful tricuspid repair for Ebstein malformation (EM) is challenging. Cone repair has recently emerged as the most anatomic repair technique. There are no data on tricuspid re-repair in EM. We reviewed our experience with tricuspid valve (TV) re-repair using a cone technique. Between June 2007 and October 2012, 134 patients underwent cone repair. Prior TV repair was performed in 20 patients (10 female, 50%). Median age was 15 years (range, 4 to 68 years). Four patients (20%) had prior bidirectional cavopulmonary anastomosis (BDCPA). Preoperative heart failure was present in 8 patients (40%). Recurrent tricuspid regurgitation (TR) was due to incomplete leaflet coaptation with tethered anterior leaflet in all patients; and 10 patients (50%) had diminutive septal leaflet. Prior repair techniques consisted of annuloplasty maneuvers in all patients with no or incomplete surgical delamination in all patients. The TV re-repair using cone technique was possible in all patients. Plication of atrialized right ventricle was done in 6 patients (30%). Modifications included annuloplasty band in 16 patients (80%) and TV leaflet augmentation in 8 (40%). The BDCPA was performed in 3 patients (15%) and modified Cox-maze procedure was done in 10 (50%). There were no mortalities or reoperations. Mean follow-up was 7.7 ± 10.7 months; during follow-up, 18 patients had no or mild TR and 2 had moderate TR. TV re-repair in EM using the cone technique is feasible, particularly when there has been no surgical leaflet delamination at initial operation. Leaflet augmentation facilitates and annuloplasty band stabilizes a competent TV re-repair. Longer follow-up is required to assess durability.


Elevated CA-125 Level and ER-Negative as Prognostic Factors for Ovarian Metastasis in Patients with Endometrial Cancer: A Retrospective Cohort Study.
BACKGROUND The utility of cancer antigen 125 (CA-125), estrogen receptor (ER), and progesterone receptor (PR) in evaluation for ovarian metastasis of endometrial cancer has yet to be determined. The purpose of this study was to investigate the incidence and the possible risk factors of ovarian metastasis. MATERIAL AND METHODS A retrospective study was performed in endometrial cancer patients who accepted surgical intervention of hysterectomy and oophorectomy during 2002-2013 in Sun Yat-sen Memorial Hospital, Sun Yat-sen University, China. Clinico-pathologic characteristics and the possible risk factors were investigated. RESULTS A total of 565 patients were identified, of which 5.7% had ovarian metastasis. Univariate analysis and multivariate analysis revealed that deeper myometrial invasion, tubal involvement, and parametrial involvement were independent risk factors. In subgroup analysis, univariate analysis showed that elevated CA-125 level and negative ER were associated with ovarian metastasis (P<0.05), however multivariate analysis revealed that only high CA-125 level was an independent risk factor (P<0.05). The incidence of ovarian metastasis in patients with high CA-125 level and who were ER-negative was 24%. For patients with normal CA-125 level and who were ER-positive, the incidence was 1.19%. The optimal cutoff value that provided the best sensitivity and specificity was 110.5 U/ml. CONCLUSIONS The incidence of ovarian metastasis in endometrial cancer is low. Ovarian preservation should be considered for women without abnormal CA-125 level and who have deeper myometrial invasion, tubal involvement, parametrial involvement, and who are ER-negative. These findings may facilitate clinical decision-making.


[Preparation and identification of monoclonal antibodies against Helicobacter pylori].
To prepare and identify monoclonal antibodies (mAbs) against Helicobacter pylori (Hp). BALB/c mice were immunized with the supernatant and precipitation of cultured Hp after ultrasonication and mAbs were obtained by means of hybridoma technique. The resultant mAbs was evaluated for subtype, titer, affinity, and further identified with Lpp20, HspA, urease A, CagA, urease B, and catalase prepared by recombinant expression. Totally 34 hybridoma cell lines were established which secreted specific mAbs, including 31 against the supernatant and 3 against the precipitation of Hp, and the prepared mAbs showed specific reaction against Lpp20 (3 strains), HspA (2 strains), urease A (4 strains), CagA (1 strain), urease B (5 strains), and catalase (2 strains) antigens, respectively. The mAbs was all identified as immunoglobulin G1 (IgG1) and theirs titer in the culture supernatant and ascites was 1:16 to 1:32 and 1:32000 to 1:64000 respectively with affinity constants (K(aff)) ranging from 1 x 10(-10) to 5.2 x 10(-12) mol/L. The mAbs specially against Hp have been obtained, which may facilitate further study of detection and vaccine development of Hp.


[Effect of high 2-KLG concentration on expression of pivotal genes involved in 2-KLG synthesis in Gluconobacter oxydans WSH-003].
To analyze the effect of high 2-keto-L-gulonic acid (2-KLG) on important dehydrogenase, cofactor and transport proteins involved in 2-KLG synthesis. First, the growth of Gluconobacter oxydans under high 2-KLG was observed. The real-time PCR was used to detect the expression of key sorbitol dehydrogenase gene sldAB, pyrroloquinoline quinone (PQQ) biosynthesis gene cluster pqqABCDE, and five genes encoding hypothetic PQQ transport proteins. According to results of the growth of G. oxydans under different 2-KLG concentration, 40, 80 and 120 g/L 2-KLG were decided to stimulate strains. Real-time PCR showed that PQQ synthesis genes pqqABCDE were not affected by high 2-KLG, but sorbitol dehydrogenase genes sldAB and part of genes encoding PQQ transport proteins were down-regulated under high 2-KLG stress. The expression of sorbitol dehydrogenase genes was restrained by high 2-KLG, PQQ transport was probably inhibited, but PQQ synthesis was not affected.


Inhibition of reactive oxygen species attenuates aneurysm formation in a murine model.
Reactive oxygen species (ROS) are increased in human abdominal aortic aneurysms (AAA). NADPH oxidases are the predominant source of superoxide anion (O(2)(-)) in the vasculature. Inducible nitric oxide synthase (iNOS) produces a significant amount of nitric oxide (NO) during inflammatory processes. We hypothesized that ROS produced by NADPH oxidases and iNOS played an important role in aneurysm formation. We examined this hypothesis using selective blockade of NADPH oxidases and iNOS in a murine model of AAA. Mice, including C57BL/6, iNOS knockout (iNOS(-/-)) mice, and its background matched control (C57BL/6), underwent AAA induction by periaortic application of CaCl(2). Aortic diameter was measured at aneurysm induction and harvest. Beginning 1 week prior to aneurysm induction and continuing to aortic harvest 6 weeks later, one group of the C57BL/6 mice were treated with orally administered apocynin (NADPH oxidase inhibitor). Control mice were given water. The mean diameter and change in diameter of each group were compared with concurrent controls. Aortic levels of the NO metabolite, NO(x) (NO(2) and NO(3)), were significantly increased in CaCl(2)-treated wild type mice. INOS(-/-) mice were partly resistant to aneurysm induction. This was associated with reduced expression of matrix metalloproteinase (MMP)-2 and MMP-9 and decreased production of NO(x) in the aortic tissues. Inhibition of NADPH oxidase by apocynin also blocked aneurysm formation. In conclusion, both iNOS deficiency and NADPH oxidase inhibition suppressed aneurysm formation in association with decreased NO(x) levels. These studies suggest that both NADPH oxidase and iNOS pathways contribute to ROS production and AAA development.


Effects of tannic acid on membrane fouling and membrane cleaning in forward osmosis.
The fouling behavior during forward osmosis (FO) was investigated. Tannic acid was used as a model organic foulant for natural organic matter analysis since the main characteristics are similar, and calcium ions were added at different concentrations to explore the anti-pollution capability of FO membranes. The initial permeate flux and calcium ions strength were varied in different operating conditions to describe membrane fouling with membrane cleaning methods. The observed flux decline in FO changed dramatically with the type of foulant and the type of draw solution used to provide the osmotic driving force. Calcium ions aggravated membrane fouling and decreased transmembrane flux. Membrane cleaning methods included physical and physicochemical approaches, and there was no obvious difference among the typical cleaning methods (i.e., membrane flushing with different types of cleaning fluids at various crossflow velocities and backwashing with varying osmotic driving forces) with respect to flux recovery. Ultrasonic cleaning damaged the membrane structure and decreased permeate flux, and reverse diffusion of salt from the draw solution to the feed side accelerated after cleaning.


A new action potential detector using the MTEO and its effects on spike sorting systems at low signal-to-noise ratios.
This paper considers neural signal processing applied to extracellular recordings, in particular, unsupervised action potential detection at a low signal-to-noise ratio. It adopts the basic framework of the multiresolution Teager energy operator (MTEO) detector, but presents important new results including a significantly improved MTEO detector with some mathematical analyses, a new alignment technique with its effects on the whole spike sorting system, and a variety of experimental results. Specifically, the new MTEO detector employs smoothing windows normalized by noise power derived from mathematical analyses and has an improved complexity by utilizing the sampling rate. Experimental results prove that this detector achieves higher detection ratios at a fixed false alarm ratio than the TEO detector and the discrete wavelet transform detector. We also propose a method that improves the action potential alignment performance. Observing that the extreme points of the MTEO output are more robust to the background noise than those of the action potentials, we use the MTEO output for action potential alignment. This brings not only noticeable improvement in alignment performance but also quite favorable influence over the classification performance. Accordingly, the proposed detector improves the performance of the whole spike sorting system. We verified the improvement using various modeled neural signals and some real neural recordings.


New antiepileptic drugs. Overcoming the limitations of traditional therapy.
Several new antiepileptic drugs have become available recently. Since seizures and epilepsy are common, primary care physicians are likely to encounter a patient who is taking one of these new medications. Successful medical management of epilepsy requires a proper understanding of medication half-life, indications, and side effects. Felbamate has a broad spectrum of efficacy but is limited by side effects and idiosyncratic reactions. Fosphenytoin has the efficacy of phenytoin and offers the advantage of intramuscular and intravenous dosing without the significant adverse effects associated with intravenous phenytoin; however, it is expensive. Gabapentin has minimal side effects and drug interactions yet has limited efficacy for seizures. Lamotrigine has broad seizure efficacy but requires a slow adjustment to therapeutic levels. Topiramate has minimal drug interactions, but therapy must be initiated slowly to avoid side effects. All of the new antiepileptics hold great promise in the management of patients with recurrent seizures.


Serum Glycoprotein Biomarker Discovery and Qualification Pipeline Reveals Novel Diagnostic Biomarker Candidates for Esophageal Adenocarcinoma.
We report an integrated pipeline for efficient serum glycoprotein biomarker candidate discovery and qualification that may be used to facilitate cancer diagnosis and management. The discovery phase used semi-automated lectin magnetic bead array (LeMBA)-coupled tandem mass spectrometry with a dedicated data-housing and analysis pipeline; GlycoSelector (http://glycoselector.di.uq.edu.au). The qualification phase used lectin magnetic bead array-multiple reaction monitoring-mass spectrometry incorporating an interactive web-interface, Shiny mixOmics (http://mixomics-projects.di.uq.edu.au/Shiny), for univariate and multivariate statistical analysis. Relative quantitation was performed by referencing to a spiked-in glycoprotein, chicken ovalbumin. We applied this workflow to identify diagnostic biomarkers for esophageal adenocarcinoma (EAC), a life threatening malignancy with poor prognosis in the advanced setting. EAC develops from metaplastic condition Barrett's esophagus (BE). Currently diagnosis and monitoring of at-risk patients is through endoscopy and biopsy, which is expensive and requires hospital admission. Hence there is a clinical need for a noninvasive diagnostic biomarker of EAC. In total 89 patient samples from healthy controls, and patients with BE or EAC were screened in discovery and qualification stages. Of the 246 glycoforms measured in the qualification stage, 40 glycoforms (as measured by lectin affinity) qualified as candidate serum markers. The top candidate for distinguishing healthy from BE patients' group was Narcissus pseudonarcissus lectin (NPL)-reactive Apolipoprotein B-100 (p value = 0.0231; AUROC = 0.71); BE versus EAC, Aleuria aurantia lectin (AAL)-reactive complement component C9 (p value = 0.0001; AUROC = 0.85); healthy versus EAC, Erythroagglutinin Phaseolus vulgaris (EPHA)-reactive gelsolin (p value = 0.0014; AUROC = 0.80). A panel of 8 glycoforms showed an improved AUROC of 0.94 to discriminate EAC from BE. Two biomarker candidates were independently verified by lectin magnetic bead array-immunoblotting, confirming the validity of the relative quantitation approach. Thus, we have identified candidate biomarkers, which, following large-scale clinical evaluation, can be developed into diagnostic blood tests. A key feature of the pipeline is the potential for rapid translation of the candidate biomarkers to lectin-immunoassays.


Randomized placebo-controlled trial of feline-origin Enterococcus hirae probiotic effects on preventative health and fecal microbiota composition of fostered shelter kittens.
Diarrhea is the second most common cause of mortality in shelter kittens. Studies examining prevention strategies in this population are lacking. Probiotics are of particular interest but studies in cats are largely limited to healthy adults or those with induced disease. Only one study in domestic cats describes the use of host-derived bacteria as a probiotic. We previously identified Enterococcus hirae as a dominant species colonizing the small intestinal mucosa in healthy shelter kittens. Oral administration of a probiotic formulation of kitten-origin E. hirae (strain 1002-2) mitigated the increase in intestinal permeability and fecal water loss resulting from experimental enteropathogenic E. coli infection in purpose-bred kittens. Based on these findings, we hypothesized that administration of kitten-origin E. hirae to weaned fostered shelter kittens could provide a measurable preventative health benefit. We conducted a randomized, placebo-controlled, blinded clinical trial to determine the impact of a freeze-dried E. hirae probiotic on body weight gain, incidence of diarrhea, carriage of potential diarrheal pathogens, and composition of the intestinal microbiota in weaned fostered shelter kittens. One-hundred thirty kittens completed the study. Fifty-eight kittens received the probiotic and 72 received the placebo. There were no significant differences in age, weight upon initiation of the study, number of days in the study, average daily gain in body weight, or weight at completion of the study. Kittens treated with E. hirae were 3.4 times less likely to develop diarrhea compared to kittens treated with placebo (odds ratio = 0.294, 95% CI 0.109-0.792, p = 0.022). A significant impact of E. hirae was not observed on the presence or abundance of 30 different bacterial, viral, protozoal, fungal, algal, and parasitic agents in feces examined by qPCR. With exception to a decrease in Megamonas, administration of the E. hirae probiotic did not alter the predominant bacterial phyla present in feces based on 16S rRNA gene amplicon sequencing. Decreased incidence of diarrhea associated with preventative administration of E. hirae to foster kittens supports a rationale for use of E. hirae for disease prevention in this young population at high risk for intestinal disease though additional studies are warranted.


Molecular mapping of the rosy locus in Drosophila melanogaster.
The DNA from the chromosomal region of the Drosophila rosy locus has been examined in 83 rosy mutant strains. Several spontaneous and radiation-induced alleles were associated with insertions and deletions, respectively. The lesions are clustered in a 4-kb region. Some of the alleles identified on the DNA map have been located on the genetic map by fine-structure recombination experiments. The genetic and molecular maps are collinear, and the alignment identifies the DNA location of the rosy control region. A rosy RNA of 4.5 kb has been identified; its 5' end lies in or near the control region.


Disseminate and recurrent infundibulofolliculitis.
Disseminate and recurrent infundibulofolliculitis is an uncommon pruitic papular follicular eruption that affects the trunk and proximal extremities and occurs almost exclusively in blacks. Little is known about the cause, and treatment has been totally unsuccessful. Extensive laboratory investigations, including histology and direct immunofluorescence in four cases, were unrevealing. Oral vitamin A therapy was effective in controlling five of six cases. We consider this disease to be a specific clinical and pathologic response to an, as yet, unknown cause or causes.


Challenging Behavior, Parental Conflict and Community Violence in Students with Aggressive Behavior.
The effects of the presence of challenging behavior problems, parental conflict and violence in the community were determined by the probability of occurrence of bullying behaviors in elementary students. 664 students participated in the study, of whom 80 (12.04%) were identified as aggressors. 80 students with no reports of attacks were later selected randomly for comparison. Using logistic regression, it was found that the variables studied manifest significant differences between the student groups with and without aggressive behavior toward peers (R2 = .39). Challenging behavior (OR = 7.83), parental conflict (OR = 3.77) and Community Violence (OR = 5.36) increase the probability of belonging to the group of aggressors. We conclude that it is necessary to analyze the bullying from an ecological framework that considers variables located in the contexts in which individuals interact.


Review article: breath testing for human liver function assessment.
Carbon-labelled breath tests were proposed as tools for the evaluation of human liver function 30 years ago, but have never become part of clinical routine. One reason for this is the complex role of the liver in metabolic regulation, making it difficult to provide essential information for the management of patients with liver disease with a single test and to satisfy the hepatology community. As a result, a battery of breath tests have been developed. Depending on the test compound administered, different metabolic pathways (microsomal, cytosolic, mitochondrial) can be examined. Most available data come from microsomal function tests, whilst information about cytosolic and mitochondrial liver function is more limited. However, breath tests have shown promise in some studies, in particular to predict the outcome of patients with chronic liver disease or to monitor hepatic function after treatment. Whilst we await new substrates that can be used to measure liver function in a more valid manner, and large prospective studies to assess the usefulness of available test compounds, the aim of this review is to describe how far we have come in this controversial and unresolved issue.


WITHDRAWN: Adenosine versus intravenous calcium channel antagonists for the treatment of supraventricular tachycardia in adults.
Patients with paroxysmal supraventricular tachycardia frequently present to the Emergency Department. Where vagal manoeuvres fail, the two most commonly used drugs are adenosine and calcium channel antagonists. Both are known to be effective but both have a significant side-effect profile. To examine the relative effects of adenosine and calcium channel antagonists and, if possible, to determine which is most appropriate for the management of supraventricular tachycardia. Studies were identified from The Cochrane Central Register of Controlled Trials (CENTRAL) on The Cochrane Library, Issue 2, 2010, MEDLINE (1966 to May Week 1 2010) and EMBASE (1980 to 2010 week 19). The searches were originally run in June 2006 and updated and re-run in May 2010. Bibliographies of identified studies were also examined. No language restrictions were applied. Randomised trials comparing adenosine and a calcium channel antagonist in patients of any age with supraventricular tachycardia, where one of the defined outcomes was reported. Outcomes of interest were: reversion rate, mortality, time to reversion, rate of relapse, major and minor adverse events, length of hospital stay and patient satisfaction. Two authors independently checked the results of searches to identify relevant studies. Dichotomous outcomes were reported as Peto Odds ratios and continuous outcomes as weighted mean differences. A total of ten trials were identified (two new trials were identified through the updated search in May 2010), all of which used verapamil as the calcium antagonist. In the pooled analysis there was no significant difference in reversion rate between the two drugs. Time to reversion was slower for verapamil than adenosine in all studies that reported this outcome, but the data were not suitable for combining. Relapse rates were higher for adenosine compared with verapamil (OR 0.25, 95% CI 0.07 to 0.99. P=0.05). Minor adverse events such as nausea, chest tightness, shortness of breath and headache were reported much more frequently in patients treated with adenosine with 10.8 % of patients reporting at least one of these events, compared with 0.6% of those treated with verapamil (OR 0.15, 95% CI 0.09 to 0.26, P<0.001). Hypotension was reported exclusively in the verapamil treatment group (4/214), and occurred in none of the patients treated with adenosine (OR 10.8, 95% CI 1.46 to 80.22, P=0.02). Adenosine and verapamil are both effective treatments for supraventricular tachycardia in the majority of patients. There is a high incidence of minor but unpleasant side effects and a greater risk of relapse in patients treated with adenosine while some patients treated with verapamil may develop significant hypotension. Patients should be fully informed of these risks prior to treatment.


Modelling genetic diseases for drug development: Hypertrophic cardiomyopathy.
Hypertrophic cardiomyopathy (HCM) is the commonest genetic cardiac disease, with a prevalence of 1/500. It is caused by over 1400 different mutations, mainly involving the genes coding for sarcomere proteins. The main pathological features of HCM are left ventricular hypertrophy, diastolic dysfunction and the increased ventricular arrhythmogenesis. Predicting the risk of heart failure and lethal arrhythmias is the most challenging clinical task for HCM patient management. Moreover, there are no disease-modifying therapies that can prevent disease progression or sudden arrhythmic death in HCM patients. In this review, we will illustrate the most advanced research models and methods that have been employed for HCM studies, including preclinical tests of novel or existing drugs, along with visionary future development based on gene editing approaches. Acknowledging the advantages and limitations of the different models, and a critical consideration of the different, often conflicting result obtained using different approaches is essential for a deep understanding of HCM pathophysiology and for obtaining meaningful information on novel treatments, in order to improve patient risk stratification and therapeutic management.


Antigenic and genetic diversity of highly pathogenic avian influenza A (H5N1) viruses isolated in Egypt.
Highly pathogenic avian influenza A virus (H5N1) has diverged antigenically and genetically since its initial detection in Asia in 1997. Viruses belonging to clade 2.2 in particular have been reported in numerous countries with the majority occurring in Egypt. Previous reports identified antigenic similarities between viruses belonging to clade 2.2. However, poultry and human viruses isolated in northern Egypt during 2007 and 2008 were found to be antigenically distinct from other clade 2.2 viruses from this country. Genetic analysis of the hemagglutinin revealed a high degree of nucleotide and amino acid divergence. The antigenic changes in Egyptian viruses isolated during 2007-08 necessitated that two of these strains be considered as potential H5N1 pre-pandemic vaccine candidates.


Changing aetiology of paediatric septic arthritis.
The management of septic arthritis in children requires the prompt administration of antibiotic therapy and the identification of the causative pathogen. In the past, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Haemophilus influenzae type b were considered the main causative agents of the disease, but a substantial fraction of presumptive joint infections remained unconfirmed by conventional bacteriologic cultures. In the last two decades, our knowledge of the aetiology of paediatric infectious arthritis has substantially changed as the result of the implementation of vaccination programmes against H. influenzae type b and pneumococci, and by the use of improved detection methods. In 1988, the inoculation of synovial fluid aspirates into blood culture vials revealed that Kingella kingae, a commensal member of the oropharyngeal microbiota, was the prime aetiology of skeletal system infections in children aged 6-48 months. The clinical presentation of K. kingae arthritis is subtle, and the disease is frequently missed by classic clinical and laboratory diagnostic criteria. Many children are afebrile, the acute phase reactants levels and the white blood cell counts in the blood and synovial fluid specimens are frequently normal, requiring a high clinical acumen. Increasing use of sensitive molecular methods in recent years, and particularly nucleic acid amplification tests that target K. kingae-specific genes, has further improved the detection of this elusive pathogen, demonstrated that it is responsible for 30-93% of all cases of septic arthritis below 4 years of age and reduced the fraction of culture-negative infections.


Evaluation of zero-echo-time attenuation correction for integrated PET/MR brain imaging-comparison to head atlas and 68Ge-transmission-based attenuation correction.
MRI does not offer a direct method to obtain attenuation correction maps as its predecessors (stand-alone PET and PET/CT), and bone visualisation is particularly challenging. Recently, zero-echo-time (ZTE) was suggested for MR-based attenuation correction (AC). The aim of this work was to evaluate ZTE- and atlas-AC by comparison to 68Ge-transmission scan-based AC. Nine patients underwent brain PET/MR and stand-alone PET scanning using the dopamine transporter ligand 11C-PE2I. For each of them, two AC maps were obtained from the MR images: an atlas-based, obtained from T1-weighted LAVA-FLEX imaging with cortical bone inserted using a CT-based atlas, and an AC map generated from proton-density-weighted ZTE images. Stand-alone PET 68Ge-transmission AC map was used as gold standard. PET images were reconstructed using the three AC methods and standardised uptake value (SUV) values for the striatal, limbic and cortical regions, as well as the cerebellum (VOIs) were compared. SUV ratio (SUVR) values normalised for the cerebellum were also assessed. Bias, precision and agreement were calculated; statistical significance was evaluated using Wilcoxon matched-pairs signed-rank test. Both ZTE- and atlas-AC showed a similar bias of 6-8% in SUV values across the regions. Correlation coefficients with 68Ge-AC were consistently high for ZTE-AC (r 0.99 for all regions), whereas they were lower for atlas-AC, varying from 0.99 in the striatum to 0.88 in the posterior cortical regions. SUVR showed an overall bias of 2.9 and 0.5% for atlas-AC and ZTE-AC, respectively. Correlations with 68Ge-AC were higher for ZTE-AC, varying from 0.99 in the striatum to 0.96 in the limbic regions, compared to atlas-AC (0.99 striatum to 0.77 posterior cortex). Absolute SUV values showed less variability for ZTE-AC than for atlas-AC when compared to 68Ge-AC, but bias was similar for both methods. This bias is largely caused by higher linear attenuation coefficients in atlas- and ZTE-AC image compared to 68Ge-images. For SUVR, bias was lower when using ZTE-AC than for atlas-AC. ZTE-AC shows to be a more robust technique than atlas-AC in terms of both intra- and inter-patient variability.


[Pulmonary hypertension in chronic nonspecific lung diseases].
Pulmonary and intracardiac hemodynamic findings are reported. They were obtained by means of catheterization of right cardiac departments and the pulmonary artery with thermodilution in 57 patients with chronic nonspecific pulmonary diseases (CNPD), under conditions of resting, rationed bicycle-ergometric exercise, hypo- and hyperoxia, and nitroglycerin administration. Changes in pulmonary and intracardiac hemodynamics which reflect the ongoing exhaustion of compensatory mechanisms in the external respiration apparatus, the pulmonary vascular system and the heart, are shown to precede the formation of right ventricular hypertrophy in obstructive CNPD A classification of secondary pulmonary hypertension, based on clinical and instrumental signs, is offered.


Multi-institutional clinical experience with the Calypso System in localization and continuous, real-time monitoring of the prostate gland during external radiotherapy.
To report the clinical experience with an electromagnetic treatment target positioning and continuous monitoring system in patients with localized prostate cancer receiving external beam radiotherapy. The Calypso System is a target positioning device that continuously monitors the location of three implanted electromagnetic transponders at a rate of 10 Hz. The system was used at five centers to position 41 patients over a full course of therapy. Electromagnetic positioning was compared to setup using skin marks and to stereoscopic X-ray localization of the transponders. Continuous monitoring was performed in 35 patients. The difference between skin mark vs. the Calypso System alignment was found to be >5 mm in vector length in more than 75% of fractions. Comparisons between the Calypso System and X-ray localization showed good agreement. Qualitatively, the continuous motion was unpredictable and varied from persistent drift to transient rapid movements. Displacements > or =3 and > or =5 mm for cumulative durations of at least 30 s were observed during 41% and 15% of sessions. In individual patients, the number of fractions with displacements > or =3 mm ranged from 3% to 87%; whereas the number of fractions with displacements > or =5 mm ranged from 0% to 56%. The Calypso System is a clinically efficient and objective localization method for positioning prostate patients undergoing radiotherapy. Initial treatment setup can be performed rapidly, accurately, and objectively before radiation delivery. The extent and frequency of prostate motion during radiotherapy delivery can be easily monitored and used for motion management.


Enhancement of Above Threshold Ionization in Resonantly Excited Helium Nanodroplets.
Clusters and nanodroplets hold the promise of enhancing high-order nonlinear optical effects due to their high local density. However, only moderate enhancement has been demonstrated to date. Here, we report the observation of energetic electrons generated by above-threshold ionization (ATI) of helium (He) nanodroplets which are resonantly excited by ultrashort extreme ultraviolet (XUV) free-electron laser pulses and subsequently ionized by near-infrared (NIR) or near-ultraviolet (UV) pulses. The electron emission due to high-order ATI is enhanced by several orders of magnitude compared with He atoms. The crucial dependence of the ATI intensities with the number of excitations in the droplets suggests a local collective enhancement effect.


[Disorder of selective attention in patients with late-manifesting schizophrenia with a syndrome of involutional paranoia].
Selective attention was studied by registering cortical evoked potentials to acoustic stimuli in mental patients and mentally healthy subjects of old age. Disorder of selective attention in the patients was manifested in an impaired ability of the higher portions of the central nervous system to differentiate between essential and non-essential external stimuli as well as in a weakened inhibition of irrelevant information.


Endothelin-1 and endothelin receptor gene variants and their association with negative outcomes following aneurysmal subarachnoid hemorrhage.
Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease that affects approximately 30,000 people a year in the United States. Delayed cerebral ischemia (DCI) and cerebral vasospasm (CV) are common complications after aSAH. In addition, aSAH patients have a high risk of poor long-term outcomes. Endothelin-1 (ET-1), a potent vasoconstrictor, or its two types of receptors, ET receptor A (ETA) and ET receptor B (ETB), may play a role in the pathogenesis of DCI and CV. Genetic variations within the ET-1, ETA, or ETB genes may also account for variance observed in the outcomes of aSAH patients. The purpose of this study was to describe the distribution of the Lys198Asn polymorphism, a known functional SNP in the ET-1 gene, and tagging SNPs of the ET-1, ETA, and ETB genes in individuals recovering from aSAH. This study also investigated the relationships among the ET polymorphisms, DCI, and global functional outcomes measured at 3 and 6 months after aSAH. Participants included individuals aged 18-75 years with a diagnosis of aSAH. There was a trend found between the variant allele of an ET-1 SNP (rs6912834) and angiographic vasospasm. There were also associations found between two ETB SNPs (rs9574124 and rs3027111) and poor outcomes as measured by the Glasgow Outcome scale at 3 months. These findings support the role of ET-1 and ETB in recovery following aSAH.


Establishing Medical Coverage and Epidemiological Surveillance during the Grand Magal of Touba in Senegal: A Public Health Need.
The Grand Magal is a religious pilgrimage that takes place in Senegal. An estimated 4-5 million individuals yearly gather in the holy city of Touba. Pilgrims comes from the whole Senegal and surrounding countries and from countries outside of Africa where Mouride Senegalese emigrated. It is the largest Mass Gathering (MG) event of the Mouride community and the largest Muslim religious MG in West Africa. The context of the Grand Magal MG is unique given its location in a tropical developing country and its international component which may favour the globalization of local endemic diseases and warrants investment in modern methods for public health surveillance and planning of the event.


Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores.
Divalent ions are known to have a severe effect on the translocation of several antibiotic molecules into (pathogenic) bacteria. In the present study we have investigated the effect of divalent ions on the permeability of norfloxacin across the major outer membrane channels from E. coli (OmpF and OmpC) and E. aerogenes (Omp35 and Omp36) at the single channel level. To understand the rate limiting steps in permeation, we reconstituted single porins into planar lipid bilayers and analyzed the ion current fluctuations caused in the presence of norfloxacin. Moreover, to obtain an atomistic view, we complemented the experiments with millisecond-long free energy calculations based on temperature-accelerated Brownian dynamics simulations to identify the most probable permeation pathways of the antibiotics through the respective pores. Both, the experimental analysis and the computational modelling, suggest that norfloxacin is able to permeate through the larger porins, i.e., OmpF, OmpC, and Omp35, whereas it only binds to the slightly narrower porin Omp36. Moreover, divalent ions can bind to negatively charged residues inside the porin, reversing the ion selectivity of the pore. In addition, the divalent ions can chelate with the fluoroquinolone molecules and alter their physicochemical properties. The results suggest that the conjugation with either pores or molecules must break when the antibiotic molecules pass the lumen of the porin, with the conjugation to the antibiotic being more stable than that to the respective pore. In general, the permeation or binding process of fluoroquinolones in porins occurs irrespective of the presence of divalent ions, but the presence of divalent ions can vary the kinetics significantly. Thus, a detailed investigation of the interplay of divalent ions with antibiotics and pores is of key importance in developing new antimicrobial drugs.


Specific Buffers Affect the Stability of a Charged Cyclodextrin Complex Via Competitive Binding and Ionic Strength.
The effect of 11 buffers as well as the effect of ionic strength were investigated on the binding between the bile salt taurochenodeoxycholate and the ionic sulfobutylether-β-cyclodextrin. The investigations showed that both ionic strength and competitive binding affected the stability constant. The stability constant for the sulfobutylether-β-cyclodextrin complex increased from 34,400 M-1 to 114,000 M-1 as the ionic strength of the solution increased to 0.15 M. Keeping the ionic strength constant, the stability constant for the complex depended on the buffer in the solution, with citric and succinic acid reducing the stability constant. The reduction in the stability constant by buffers was related to a competitive mechanism. The results showed that, when accounting for the variation in ionic strength between the buffers, three groupings of buffers existed. All carboxylic acid buffers decreased the stability constant of the sulfobutylether-β-cyclodextrin complex, relative to the effect observed by altering the ionic strength, whereas the other buffers only affected the stability constant in terms of the changes in ionic strength. Both buffer species and ionic strength impacted the stability of ionic cyclodextrin complexes, hence, it is important to be aware of these effects when working with, comparing and reporting stability constants.


Traditional Chinese Medicine Tanreqing Targets Both Cell Division and Virulence in Staphylococcus aureus.
Staphylococcus aureus has been recognized as an important human pathogen and poses a serious health threat worldwide. With the advent of antibiotic resistance, such as the increased number of methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to develop new therapeutical agents. In this study, Chinese traditional medicine Tanreqing (TRQ) has been used as an alternative treating agent against MRSA and we aim to unravel the mode of action of TRQ underlying MRSA inhibition. TRQ treatment affected numerous gene expression as revealed by RNA-seq analysis. Meanwhile, TRQ targeted cell division to inhibit cell growth as shown by illumination microscopy. Besides, we confirmed that TRQ downregulates the expression of virulence factors such as hemolysin and autolysin. Finally, we used a murine model to demonstrate that TRQ efficiently reduces bacterial virulence. Altogether, we have proved TRQ formula to be an effective agent against S. aureus infections.


Endothelial progenitor and mesenchymal stem cell-derived cells persist in tissue-engineered patch in vivo: application of green and red fluorescent protein-expressing retroviral vector.
An unresolved question regarding tissue-engineered (TE) cardiac valves and vessels is the fate of the transplanted cells in vivo. We have developed a strategy to track the anatomic location of seeded cells within TE constructs and neighboring tissues using a retroviral vector system encoding green and red fluorescent proteins (GFPs and RFPs, respectively) in ovine circulating endothelial progenitor cells (EPCs) and bone marrow-derived mesenchymal stem cells (BMSCs). We demonstrate that stable transduction ex vivo with high-titer Moloney murine leukemia virus-based retroviral vector yields transduction efficiency of greater than 97% GFP(+) EPC- and RFP(+) mesenchymal stem cell (MSC)-derived cells. Cellular phenotype and transgene expression were also maintained through 25 subsequent passages. Using a retroviral vector system to distinguish our pre-seeded cells from tissue-resident progenitor cells and circulating endothelial and marrow-derived precursors, we simultaneously co-seeded 2 x 10(6) GFP(+) EPCs and 2 x 10(5) RFP(+) MSCs onto the TE patches. In a series of ovine pulmonary artery patch augmentation studies, transplanted GFP(+) EPC- and RFP(+) MSC-derived cells persisted within the TE patch 7 to 14 days after implantation, as identified using immunofluorescence. Analysis showed 81% luminal coverage of the TE patches before implantation with transduced cells, increasing to 96% at day 7 and decreasing to 67% at day 14 post-implantation. This suggests a temporal association between retroviral expression of progenitor cells and mediating effects of these cells on the physiological remodeling and maturation of the TE constructs. To our knowledge, this is the first cardiovascular tissue-engineering in vivo study using a double-labeling method to demonstrate a direct evidence of the source, persistence, and incorporation into a TE vascular patch of co-cultured and simultaneously pre-seeded adult progenitor cells.


Identification of an enzyme in human kidney that correctly processes prorenin.
Using pure recombinant human prorenin as a substrate, we have identified an enzyme in human kidney that accurately processes prorenin to active renin (EC 188.8.131.52). In the crude homogenate, the predominant activity of this potential renin-processing enzyme (RPE) converted the Mr 47,000 inactive prorenin to Mr 44,000 active renin and had a pH optimum of approximately 6. The activity was blocked by cysteine protease inhibitors, but not by pepstatin, EDTA, or serine protease inhibitors. This RPE activity was not detected in a similarly prepared homogenate of human chorion decidua tissue, which produces primarily prorenin, or in human plasma. The activity was purified 100-fold by ammonium sulfate precipitation, p-chloromercuribenzoate affinity chromatography, and chromatofocusing. The partially purified enzyme has a Mr of approximately 27,000 and an isoelectric point in the pH 4.8-5.6 range. The activity in the purified RPE preparation had the same pH optimum as that in crude homogenate, cleaved the prosegment at the same site used by the kidney in vivo based on amino-terminal sequencing of the processed renin, and did not degrade prorenin or renin. These data suggest that the cysteine protease we have isolated is a candidate for authentic renal RPE.


Computing body segment trajectories in the Hybrid III dummy using linear accelerometer data.
An analytical method was developed and tested using several mini-sled and Hyge sled tests to calculate the planar trajectory of a Hybrid III dummy head. Aimed at expediting the Hybrid III test analyses, it may provide an opportunity for cost savings through reduced hardware and manpower on film analyses. Transformation from the moving coordinate to the laboratory coordinate is based on the angular positions integrated from the derived angular accelerations. Gravitational correction of the linear accelerometers was found to be insignificant. The computed head trajectories were compared to the ones obtained from the high speed film images. Accuracy of the calculated head trajectory relies heavily on the accuracy of the computed angular acceleration. Strain-gaged accelerometers are not dependable at all times during an impact and an ill-behaved signal for a very short period may create a significant drift in computed displacement due to double integrations. Accuracy of the currently available accelerometers is not high enough for an angular displacement calculation. A new generation of accelerometers with higher accuracy, or an angular velocity sensor may provide more accurate angular displacement for trajectory analyses. The redundancy of the in-line accelerations helps improve the isolation of erroneous outputs and improve accuracy of the procedure.


Longitudinal evaluation of cerebral morphological changes in Parkinson's disease with and without dementia.
To investigate the pattern of brain atrophy across time in a sample of Parkinson's disease (PD) patients with and without dementia using voxelbased morphometry (VBM) analysis. The initial sample comprised thirteen non-demented PD patients and sixteen demented patients. Longitudinal cognitive assessment and structural MRI were performed. The mean follow-up period was 25 months (SD=5.2). From this initial group, eight PD patients with dementia (5 men and 3 women) and eleven PD patients without dementia (7 men and 4 women) were reevaluated. MRI 3D structural images were acquired and analyzed by means of the optimized VBM procedure with Statistical Parametric Mapping (SPM2). VBM analysis showed a progressive grey matter volume decrease in patients with PD without dementia in limbic, paralimbic and neocortical associative temporooccipital regions. In patients with dementia the loss mainly involved neocortical regions. VBM revealed a significant loss of grey matter volume in PD patients with and without dementia with disease progression. The decrease in limbic and paralimbic regions is widespread in non-demented patients. Neocortical volume reduction is the most relevant finding in patients with dementia. This suggests that the neocortex is a substrate for dementia in Parkinson disease.


CDX-2 Expression in Primary Lung Adenocarcinoma.
Adenocarcinoma with enteric differentiation is a rare subtype of lung adenocarcinoma that is recognized as a variant type of primary adenocarcinoma in the 2015 World Health Organization classification of lung tumors. Published immunohistochemistry studies show variable staining pattern for CDX-2 ranging from positivity in 71% of the cases to no staining. As little is known about CDX-2 expression in lung adenocarcinomas lacking histologic features of enteric differentiation, our aim was to determine the rate of CDX-2 positivity in non-enteric-type lung adenocarcinomas. We performed immunohistochemistry for CDX-2, CK7, CK20, TTF-1, napsin A, and p40 using 4-μm sections of a previously constructed tissue microarray containing 93 primary lung adenocarcinomas that lack morphologic evidence of enteric differentiation. The cohort included 22 well, 54 moderately, and 17 poorly differentiated tumors (55 female, 38 male; age range: 42 to 86, median: 64.5). All 93 tumors were strongly CK7 positive, whereas variable CK20 staining was seen in 4 tumors (1 strong, 1 moderate, and 2 focal). Both TTF-1 and napsin A were positive in 81 of 93 (87%) tumors with only 6 of 93 (6.5%) tumors negative for both the markers. Eleven tumors were CDX-2 positive (5 strong, 3 moderate, and 3 weak), all of which were also TTF-1 and napsin A positive and p40 negative. One CDX-2-positive tumor showed focal CK20 staining. Mutation studies for EGFR/K-ras/ALK were performed in four CDX-2-positive tumors and detected a K-ras mutation in one of them. CDX-2 positivity can be seen in a subset (12%) of lung adenocarcinoma. These tumors are CK7, TTF-1, and napsin A positive and p40 negative. Focal CK20 staining is only seen in rare cases. CDX-2 positivity should not be used as the only criteria to exclude lung origin.


Transcriptional analysis of the acid tolerance response in Streptococcus pneumoniae.
Streptococcus pneumoniae, one of the major causes of morbidity and mortality in humans, faces a range of potentially acidic conditions in the middle and late stages of growth in vitro, in diverse human fluids during the infection process, and in biofilms present in the nasopharynx of carriers. S. pneumoniae was shown to develop a weak acid tolerance response (ATR), where cells previously exposed to sublethal pHs (5.8-6.6) showed an increased survival rate of up to one order of magnitude after challenge at the lethal pH (4.4, survival rate of 10(-4)). Moreover, the survival after challenge of stationary phase cells at pH 4.4 was three orders of magnitude higher than that of cells taken from the exponential phase, due to the production of lactic acid during growth and increasing acidification of the growth medium until stationary phase. Global expression analysis after short-term (5, 15 and 30 min, the adaptation phase) and long-term (the maintenance phase) acidic shock (pH 6.0) was performed by microarray experiments, and the results were validated by real-time RT-PCR. Out of a total of 126 genes responding to acidification, 59 and 37 were specific to the adaptation phase and maintenance phase, respectively, and 30 were common to both periods. In the adaptation phase, both up- and down-regulation of gene transcripts was observed (38 and 21 genes, respectively), whereas in the maintenance phase most of the affected genes were down-regulated (34 out of 37). Genes involved in protein fate (including those involved in the protection of the protein native structure) and transport (including transporters of manganese and iron) were overrepresented among the genes affected by acidification, 8.7 and 24.6 % of the acid-responsive genes compared to 2.8 % and 9.6 % of the genome complement, respectively. Cross-regulation with the response to oxidative and osmotic stress was observed. Potential regulatory motifs involved in the ATR were identified in the promoter regions of some of the regulated genes.


Characterization of DNA adducts formed by the four configurationally isomeric 5,6-dimethylchrysene 1,2-dihydrodiol 3,4-epoxides.
The DNA adducts formed from the racemic syn and anti dihydrodiol epoxides of 5,6-dimethylchrysene were characterized through various spectroscopic methods. Substantial reaction with the amino groups of both deoxyadenosine and deoxyguanosine residues were detected with both the syn and anti derivatives. The chemical shifts and coupling constants for the cis and trans opened adducts from the syn dihydrodiol epoxide were distinctly different, whereas for the anti dihydrodiol epoxide these properties were fairly similar for cis and trans adducts. In the latter case, assignment of trans and cis configurations was less obvious, and the finding that trans adducts have always predominated over cis adducts for all dihydrodiol epoxides studied to date was helpful in making these assignments. The preferential formation of cis adducts in DNA by the syn dihydrodiol epoxide is more like the chemistry of the dihydrodiol epoxide of benzo[c]phenanthrene than of benzo[g]chrysene, although both of these, like 5,6-dimethylchrysene, are non-planar compounds.


Ancestral diterpene cyclases show increased thermostability and substrate acceptance.
Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7-13 °C. Ancestral spiroviolene synthases catalyze cyclization of the natural C20 -substrate geranylgeranyl diphosphate (GGPP) and also accept C15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate-rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., kcat /KM for GGPP over kcat /KM for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.


High level in vivo mucin-type glycosylation in Escherichia coli.
Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.


NDVI to Detect Sugarcane Aphid Injury to Grain Sorghum.
Multispectral remote sensing has potential to provide quick and inexpensive information on sugarcane aphid, Melanaphis sacchari (Zehntner), pest status in sorghum fields. We describe a study conducted to determine if injury caused by sugarcane aphid to sorghum plants in fields of grain sorghum could be detected using multispectral remote sensing from a fixed wing aircraft. A study was conducted in commercial grain sorghum fields in the Texas Gulf Coast region in June 2014. Twenty-six commercial grain sorghum fields were selected and rated for the level of injury to sorghum plants in the field caused by sugarcane aphid. Plant growth stage ranged from 5.0 (watery ripe) to 7.0 (hard dough) among fields; and plant injury rating from sugarcane aphid ranged from 1.0 (little or no injury) to 4.0 (>40% of plants displaying injury) among fields. The normalized differenced vegetation index (NDVI) is calculated from light reflectance in the red and near-infrared wavelength bands in multispectral imagery and is a common index of plant stress. High NDVI indicates low levels of stress and low NDVI indicates high stress. NDVI ranged from -0.07 to 0.26 among fields. The correlation between NDVI and plant injury rating was negative and significant, as was the correlation between NDVI and plant growth stage. The negative correlation of NDVI with injury rating indicated that plant stress increased with increasing plant injury. Reduced NDVI with increasing plant growth probably resulted from reduced photosynthetic activity in more mature plants. The correlation between plant injury rating and plant growth stage was positive and significant indicating that plant injury from sugarcane aphid increased as plants matured. The partial correlation of NDVI with plant injury rating was negative and significant indicating that NDVI decreased with increasing plant injury after adjusting for its association with plant growth stage. We demonstrated that remotely sensed imagery acquired from grain sorghum fields using an airborne multi-spectral imaging system was sensitive to injury to sorghum plants caused by sugarcane aphid.


Investigations on prophylactic effects of coarse feed structure and/or potassium diformate on the microflora in the digestive tract of weaned piglets experimentally infected with Salmonella Derby.
The purpose of this study was to examine potential prophylactic effects of a coarse feed structure and/or potassium diformate (KDF as feed additive) addition to diets on the microflora of the digestive tract in reared piglets experimentally infected with Salmonella Derby. The results show that coarse grinding as well as KDF addition are able to influence positively the intestinal flora and are capable to reduce Salmonella excretion of infected piglets. Coarse grinding of main ingredients (e.g. cereals) led to an increased number of lactobacilli as well as to higher counts of Gram-positive coccoid bacteria in the colon chyme, while KDF resulted in a tendency towards lower counts of Escherichia coli within the digestive tract. Moreover, a combination of both treatments influenced the composition of the gastrointestinal flora quite positively. Furthermore, the combination of these dietetic measures resulted in a reduced Salmonella excretion rate, shorter Salmonella shedding period and a reduced translocation of Salmonella within the infected piglets. The positive effects of combining both treatments led to a significantly reduced spreading of Salmonella within the group of pigs, which might be used to diminish Salmonella prevalence in pig production.


Ursodeoxycholic acid in patients with ulcerative colitis and primary sclerosing cholangitis for prevention of colon cancer: a meta-analysis.
Colon cancer risk is high in patients with ulcerative colitis (UC) and primary sclerosing cholangitis (PSC). Ursodeoxycholic acid has been shown to have some promise as a chemopreventive agent. A meta-analysis was performed to compare the efficacy of ursodeoxycholic acid in the prevention of colonic neoplasia in patients with UC and PSC. Multiple databases were searched (January 2011). Studies examining the use of ursodeoxycholic acid vs. no ursodeoxycholic acid or placebo in adult patients with UC and PSC were included. Data were extracted in standard forms by two independent reviewers. Meta-analysis for the effect of ursodeoxycholic acid was performed by calculating pooled estimates of adenoma or colon cancer formation by odds ratio (OR) with random effects model. Heterogeneity was assessed by calculating the I (2) measure of inconsistency. RevMan 5 was utilized for statistical analysis. Four studies (n = 281) met the inclusion criteria. The studies were of adequate quality. Ursodeoxycholic acid demonstrated no overall improvement in adenoma (OR 0.53; 95 % CI: 0.19-1.48, p = 0.23) or colon cancer occurrence (OR 0.50; 95 % CI: 0.18-1.43, p = 0.20) as compared to no ursodeoxycholic acid or placebo in patients with UC and PSC. Ursodeoxycholic acid use in patients with UC and PSC does not appear to decrease the risk of adenomas or colon cancer.