CRISPR-based diagnostics detects invasive insect pests.
Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real-time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross-contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.


A tridimensional atlas of the developing human head.
The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.


Three-dimensional kinematics of the lumbar spine during gait using marker-based systems: a systematic review.
To review the current scientific literature on the assessment of three-dimensional movement of the lumbar spine with a focus on the utilisation of a 3D cluster. Electronic databases PubMed, OVID, CINAHL, The Cochrance Library, ScienceDirect, ProQuest and Web of Knowledge were searched between 1966 and March 2015. The reference lists of the articles that met the inclusion criteria were also searched. From the 1530 articles identified through an initial search, 16 articles met the inclusion criteria. All information relating to methodology and kinematic modelling of the lumbar segment along with the outcome measures were extracted from the studies identified for synthesis. Guidelines detailing 3D cluster construction were limited in the identified articles and the lack of information presented makes it difficult to assess the external validity of this technique. Scarce information was presented detailing time-series angle data of the lumbar spine during gait. Further developments of the 3D cluster technique are required and it is essential that the authors provide clear instruction, definitions and standards in their manuscript to improve clarity and reproducibility.


Development and Preliminary Evaluation of a Visual Annotation Tool to Rapidly Collect Expert-Annotated Weight Errors in Pediatric Growth Charts.
Patient weights can be entered incorrectly into electronic health record (EHR) systems. These weight errors can cause significant patient harm especially in pediatrics where weight-based dosing is pervasively used. Determining weight errors through manual chart reviews is impractical in busy clinics, and current EHR alerts are rudimentary. To address these issues, we seek to develop an advanced algorithm to detect weight errors using supervised machine learning techniques. The critical first step is to collect labelled weight errors for algorithm training. In this paper, we designed and preliminarily evaluated a visual annotation tool using Agile software development to achieve the goal of supporting the rapid collection of expert-annotated weight errors. The design was based on the fact that weight errors are infrequent and medical experts can easily spot potential errors. The results show positive user feedback and prepared us for the formal user-centered evaluation as the next step.


Colonization Rate of Potential Neonatal Disease-Causing Bacteria, Associated Factors, and Antimicrobial Susceptibility Profile Among Pregnant Women Attending Government Hospitals in Hawassa, Ethiopia.
Vaginal colonization with some species of bacteria during the last term of pregnancy can affect the health of fetuses and newborns resulting in high morbidity and mortality among newborns. The aim of this study was to determine the colonization rate of potential neonatal disease-causing bacteria, factors associated with colonization rate, and the antimicrobial susceptibility profile of bacteria among pregnant women. Institution-based cross-sectional study was conducted on pregnant women from October 13 to December 28, 2020, at government hospitals located in Hawassa, Ethiopia. Background data were captured using a structured questionnaire. Vaginal swabs were collected to isolate bacteria using the standard method. Antimicrobial susceptibility test was performed using the modified Kirby-Bauer disc diffusion method. Data were analyzed using SPSS. Factors that could predict vaginal colonization with potential neonatal disease-causing bacteria were determined using logistic regression. Overall bacterial colonization rate among pregnant women was 271 (98.9%) 95 CI (97.4‒100.1). The prevalence of potential neonatal disease-causing bacteria was 95 (34.7%) 95 CI (28.8‒40.1). The proportion of potential neonatal disease-causing bacteria were as follows: Escherichia coli (n=82, 29.9%), Acinetobacter species (n=9, 3.3%), Staphylococcus aureus (n=7. 2.6%), and Klebsiella pneumoniae (n=4, 1.5%). Pregnant women with a gestational age of 38‒40 weeks were 1.9 times (AOR= 1.9, 95% CI= 1.0-3.4, p=0.04) were more likely to be colonized by potential neonatal disease-causing bacteria. All E. coli, Klebsiella species, and Acinetobacter species were susceptible to gentamicin and imipenem. All S. aureus were susceptible to penicillin, tetracycline, clindamycin, and erythromycin. High proportion of pregnant women in this study were colonized with potential neonatal disease-causing bacteria. E. coli was the predominant bacteria. Most bacteria isolated in this study were susceptible to antimicrobial agents tested. Gestational age was significantly associated with the colonization rate of potential neonatal disease-causing bacteria.


A multicenter randomized controlled trial of medium-chain triglyceride dietary supplementation on epilepsy in dogs.
Medium-chain triglyceride (MCT) enriched diet has a positive effect on seizure control and behavior in some dogs with idiopathic epilepsy (IE). To evaluate the short-term efficacy of MCTs administered as an add-on dietary supplement (DS) to a variable base diet to assess seizure control and antiseizure drug's (ASD) adverse effect profiles. Twenty-eight dogs with International Veterinary Epilepsy Task Force Tier II (IVETF) level diagnosis of treated IE with 3 or more seizures in the last 3 months were used. A 6-month multicenter, prospective, randomized, double-blinded, placebo-controlled crossover trial was completed, comparing an MCT-DS with a control-DS. A 9% metabolic energy-based amount of MCT or control oil was supplemented to the dogs' diet for 3 months, followed by a control oil or MCT for another 3 months, respectively. Dogs enrolled in this study satisfied most requirements of IE diagnosis stated by the IVETF II level. If they received an oil DS or drugs that could influence the metabolism of the investigated DS or chronic ASD, the chronic ASD medication was adjusted, or other causes of epilepsy were found, the dogs were excluded from the study. Seizure frequency (median 2.51/month [0-6.67] versus 2.67/month [0-10.45]; P = .02) and seizure-day frequency were significantly (1.68/month [0-5.60] versus 1.99/month [0-7.42], P = .01) lower when dogs were fed MCT-DS in comparison with the control-DS. Two dogs were free of seizures, 3 had ≥50% and 12 had <50% reductions in seizure frequency, and 11 dogs showed no change or an increase in seizure frequency. These data show antiseizure properties of an MCT-DS compared to a control oil and support former evidence for the efficacy of MCTs as a nutritive, management option for a subpopulation of drug-resistant dogs with epilepsy.


Genomic signatures of convergent adaptation to Alpine environments in three Brassicaceae species.
It has long been discussed to what extent related species develop similar genetic mechanisms to adapt to similar environments. Most studies documenting such convergence have either used different lineages within species or surveyed only a limited portion of the genome. Here, we investigated whether similar or different sets of orthologous genes were involved in genetic adaptation of natural populations of three related plant species to similar environmental gradients in the Alps. We used whole-genome pooled population sequencing to study genome-wide SNP variation in 18 natural populations of three Brassicaceae (Arabis alpina, Arabidopsis halleri, and Cardamine resedifolia) from the Swiss Alps. We first de novo assembled draft reference genomes for all three species. We then ran population and landscape genomic analyses with ~3 million SNPs per species to look for shared genomic signatures of selection and adaptation in response to similar environmental gradients acting on these species. Genes with a signature of convergent adaptation were found at significantly higher numbers than expected by chance. The most closely related species pair showed the highest relative over-representation of shared adaptation signatures. Moreover, the identified genes of convergent adaptation were enriched for nonsynonymous mutations, suggesting functional relevance of these genes, even though many of the identified candidate genes have hitherto unknown or poorly described functions based on comparison with Arabidopsis thaliana. We conclude that adaptation to heterogeneous Alpine environments in related species is partly driven by convergent evolution, but that most of the genomic signatures of adaptation remain species-specific.


Experimental testing of Mackay's model for functional antagonism in the isolated costo-uterus of the rat.
Several key predictions of a recently developed model for functional antagonism (Mackay, 1981) were experimentally tested using the rat isolated costo-uterine preparation. In the presence of the functional antagonist fenoterol (Fen), the functional constants (KAF) for carbachol and oxotremorine (Oxo) were respectively 9.9 and 3.4 fold greater than their corresponding affinity constants (KA). According to Mackay's model for functional antagonism, the higher KAF/KA ratio for carbachol indicates that this cholinoceptor agonist has a greater efficacy than Oxo. This was confirmed by using conventional pharmacological methods. As predicted from the model of functional antagonism, the plot of KAF/KA-1 against the fraction of cholinoceptors not irreversibly blocked by phenoxybenzamine (Pbz) was linear for both carbachol and Oxo and the lines of best fit crossed the axes at a point not significantly different from the origin. The value of 4.6 for the relative efficacy of carbachol to Oxo estimated from functional antagonism studies was comparable to the value of 5.6 calculated using the method of irreversible antagonism proposed by Furchgott (1966).


Trust and the regulation of pharmaceuticals: South Asia in a globalised world.
Building appropriate levels of trust in pharmaceuticals is a painstaking and challenging task, involving participants from different spheres of life, including producers, distributors, retailers, prescribers, patients and the mass media. Increasingly, however, trust is not just a national matter, but involves cross-border flows of knowledge, threats and promises. Data for this paper comes from the project 'Tracing Pharmaceuticals in South Asia', which used ethnographic fieldwork and qualitative interviews to compared the trajectories of three pharmaceuticals (Rifampicin, Oxytocin and Fluoxetine) from producer to patient in three sites (north India, West Bengal and Nepal) between 2005-08. We argue that issues of trust are crucial in reducing the likelihood of appropriate use of medicines. Unlike earlier discussions of trust, we suggest that trust contexts beyond the patient-practitioner relationship are important. We illustrate these arguments through three case studies: (i) a conflict over ethics in Nepal, involving a suggested revised ethical code for retailers, medical representatives, producers and prescribers; (ii) disputes over counterfeit, fake, substandard and spurious medicines, and quality standards in Indian generic companies, looking particularly at the role played by the US FDA; and (iii) the implications of lack of trust in the DOTS programmes in India and Nepal for the relationships among patients, government and the private sector. We conclude that the building of trust is a necessary but always vulnerable and contingent process. While it might be desirable to outline steps that can be taken to build trust, the range of conflicting interests in the pharmaceutical field make feasible solutions hard to implement.


Safety, usability, and independence for wheelchair-seated drivers and front-row passengers of private vehicles: a qualitative research study.
A survey and observational study was conducted with 29 people who remain seated in their wheelchair when driving (21) or riding as a front-row passenger (8) in their personal vehicle. Each subject was observed and surveyed in their own personal vehicle that has been modified for use by occupants seated in wheelchairs. Our survey obtained responses on issues related to occupant restraint (seat belt) system usage, wheelchair securement device usage, and perception of personal safety while riding in a vehicle. Usability and accessibility issues related to seat belt and automated (docking) wheelchair securement technology were revealed, suggesting that wheelchair-seated occupants travel with a higher risk of serious injury in vehicle crashes than front-row occupants seated in original equipment manufacturer (OEM) vehicle seats and using OEM seat belts. Study results also indicate the need for improved torso support for many wheelchair-seated drivers to maintain a posture that allows for effective vehicle control. Study results demonstrate the need for innovative passive restraint technologies that provide postural support during normal vehicle operation and improved occupant restraint during crash conditions for people who drive while seated in their wheelchairs.


Infrared Spectra of the 1-Chloromethyl-1-methylallyl and 1-Chloromethyl-2-methylallyl Radicals Isolated in Solid para-Hydrogen.
The reaction of chlorine atoms (Cl) with isoprene (2-methyl-1,3-butadiene, C5H8) in solid para-hydrogen (p-H2) matrices at 3.2 K was studied using infrared (IR) spectroscopy. Mixtures of C5H8 and Cl2 were codeposited in p-H2 at 3.2 K, followed by irradiation with ultraviolet light at 365 nm to induce the photodissociation of Cl2 and the subsequent reaction of the Cl atoms with C5H8. Upon 365 nm photolysis, a multitude of new lines appeared in the IR spectrum, and, based on the secondary photolysis behavior, it was determined that the majority of the new lines belong to two distinct chemical species, designated as set A (intense lines at 1237.9, 807.8, and 605.6/608.2 cm-1, and several other weaker lines) and set B (intense lines at 942.4, 1257.7, 796.7/798.5, 667.9, and 569.7 cm-1, and several other weaker lines). Quantum-chemical calculations were performed at the B3PW91/6-311++G(2d,2p) level for ·C5H7 and the four possible isomers of the ·C5H8Cl radicals, produced from the addition of the Cl atom to the four distinct sites of carbon atoms in C5H8, to determine the relative energetics and predict IR spectra for each radical. The newly observed lines of sets A and B are assigned to the 1-chloromethyl-2-methylallyl radical (addition to carbon 4) and the 1-chloromethyl-1-methylallyl radical (addition to carbon 1) according to comparison with predicted IR spectra of possible products. The 1-chloromethyl-2-methylallyl radical and 1-chloromethyl-1-methylallyl radicals were predicted to be the most stable, with the latter ∼8 kJ mol-1 lower in energy than the former. The ratio of the 1-chloromethyl-1-methylallyl to the 1-chloromethyl-2-methylallyl radicals is estimated to be (1.2 ± 0.5):1.0, indicating that the two radicals are produced in approximately equal amounts. The exclusive production of the radicals involving the addition of the Cl atom to the two terminal carbons of isoprene is analogous to what was previously observed for the reaction of Cl atoms with trans-1,3-butadiene in solid p-H2.


Neurosurgical management of symptomatic thoracic spinal ossification in a patient with fibrodysplasia ossificans progressiva.
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by heterotopic ossification of soft connective and muscle tissues, often as the result of minor trauma. The sequelae include joint fusion, accumulation of calcified foci within soft tissues, thoracic insufficiency syndrome, and progressive immobility. The authors report on a patient with FOP who developed severe spinal canal stenosis in the thoracic spine causing substantial myelopathy. He underwent a thoracic laminectomy and resection of a large posterior osteophyte. Unique considerations are required in treating patients with FOP, including steroid administration to prevent ossification and anesthetic technique. The nuances of neurosurgical and medical management as they pertain to this disease are discussed.


Docking, Molecular Dynamics Simulation and Synthesis of New Fenobam Analogues as mGlu5 Receptor Antagonists.
Fenobam is a non-competitive mGluR5 antagonist as an anxiolytic agent. In this research a new series of fenobam analogues containing thiazole moiety instead of imidazole ring were designed and synthesized. The ureido-substituted products were synthesized from reaction of amino thiazole derivatives and isocyanate derivatives in dichloromethane solvent under microwave and ultrasonic irradiation condition. The synthesized compounds structures were established by means of IR, 1HNMR, 13CNMR spectroscopic data. Then, docking calculations were performed on the active site of mGLuR5 and compared to Fenobam as a reference drug by using AutoDock program. The molecular dynamics (MD) simulations were done using GROMACS 5.0.5. Docking studies suggested that all of the compounds possess better binding energy when compared to fenobam. The results of MD simulations might offer the binding mode of ligand (3b), accuracy of docking and the reliability of active conformations which obtained by AutoDock. New derivatives of fenobam were designed and synthesized that have the better insilico results compared to fenobam and will evaluate in future studies.


Temporal trends of persistent organic pollutants: a comparison of different time series models.
We analyzed the vapor phase atmospheric concentrations of representative persistent chemicals (i.e., α- and γ-hexachlorocyclohexane, phenanthrene, PCB-18 and PCB-52) in samples collected at a remote site near Eagle Harbor, Michigan, and at an urban site in Chicago, Illinois, using four time series models: a modified Clausius-Clapeyron equation, a multiple linear regression that includes both a linear and an harmonic dependence on time, digital filtration (DF), and dynamic harmonic regression (DHR). The results of these different models were evaluated in terms of goodness-of-fit, long-term trends, and halving times. The four approaches all provided highly significant descriptions of the data, with coefficients of determination (R(2)) ranging from 0.33 to 0.96. In general, the DF and DHR methods fit the data better, capturing not only the seasonal variations of the atmospheric concentrations but also smaller scale interannual variations in the long term trends. The halving times calculated using the four methods were generally similar to one another, and they ranged from about 4 years for γ-HCH at Chicago to about 60 years for PCB-52 at Chicago. This analysis showed that each of these four statistical methods for evaluating long-term time series has advantages and disadvantages. The choice of the appropriate method should depend on the output needed, the type of audience, and the availability and usability of the necessary software.


Revisiting the missing protein-coding gene catalog of the domestic dog.
Among mammals for which there is a high sequence coverage, the whole genome assembly of the dog is unique in that it predicts a low number of protein-coding genes, ~19,000, compared to the over 20,000 reported for other mammalian species. Of particular interest are the more than 400 of genes annotated in primates and rodent genomes, but missing in dog. Using over 14,000 orthologous genes between human, chimpanzee, mouse rat and dog, we built multiple pairwise synteny maps to infer short orthologous intervals that were targeted for characterizing the canine missing genes. Based on gene prediction and a functionality test using the ratio of replacement to silent nucleotide substitution rates (d(N)/d(S)), we provide compelling structural and functional evidence for the identification of 232 new protein-coding genes in the canine genome and 69 gene losses, characterized as undetected gene or pseudogenes. Gene loss phyletic pattern analysis using ten species from chicken to human allowed us to characterize 28 canine-specific gene losses that have functional orthologs continuously from chicken or marsupials through human, and 10 genes that arose specifically in the evolutionary lineage leading to rodent and primates. This study demonstrates the central role of comparative genomics for refining gene catalogs and exploring the evolutionary history of gene repertoires, particularly as applied for the characterization of species-specific gene gains and losses.


Formation of furan in baby food products: Identification and technical challenges.
Furan is generally produced during thermal processing of various foods including baked, fried, and roasted food items such as cereal products, coffee, canned, and jarred prepared foods as well as in baby foods. Furan is a toxic and carcinogenic compound to humans and may be a vital hazard to infants and babies. Furan could be formed in foods through thermal degradation of carbohydrates, dissociation of amino acids, and oxidation of polyunsaturated fatty acids. The detection of furan in food products is difficult due to its high volatility and low molecular weight. Headspace solid-phase microextraction coupled with gas chromatography/mass spectrometer (GC/MS) is generally used for analysis of furan in food samples. The risk assessment of furan can be characterized using margin of exposure approach (MOE). Conventional strategies including cooking in open vessels, reheating of commercially processed foods with stirring, and physical removal using vacuum treatment have remained unsuccessful for the removal of furan due to the complex production mechanisms and possible precursors of furan. The innovative food-processing technologies such as high-pressure processing (HPP), high-pressure thermal sterilization (HPTS), and Ohmic heating have been adapted for the reduction of furan levels in baby foods. But in recent years, only HPP has gained interest due to successful reduction of furan because of its nonthermal mechanism. HPP-treated baby food products are commercially available from different food companies. This review summarizes the mechanism involved in the formation of furan in foods, its toxicity, and identification in infant foods and presents a solution for limiting its formation, occurrence, and retention using novel strategies.


Cardiac and coronary function in the Langendorff-perfused mouse heart model.
The Langendorff mouse heart model is widely employed in studies of myocardial function and responses to injury (e.g. ischaemia). Nonetheless, marked variability exists in its preparation and functional properties. We examined the impact of early growth (8, 16, 20 and 24 weeks), sex, perfusion fluid [Ca(2+)] and pacing rate on contractile function and responses to 20 min ischaemia followed by 45 min reperfusion. We also assessed the impact of strain, and tested the utility of the model in studying coronary function. Under normoxic conditions, hearts from 8-week-old male C57BL/6 mice (2 mm free perfusate [Ca(2+)], 420 beats min(-1)) exhibited 145 +/- 2 mmHg left ventricular developed pressure (LVDP). Force development declined by approximately 15% (126 +/- 5 mmHg) with a reduction in free [Ca(2+)] to 1.35 mm, and by 25% (108 +/- 3 mmHg) with increased pacing to 600 beats min(-1). While elevated heart rate failed to modify ischaemic outcome, the lower [Ca(2+)] significantly improved contractile recovery (by >30%). We detected minimal sex-dependent differences in normoxic function between 8 and 24 weeks, although age modified contractile function in males (increased LVDP at 24 versus 8 weeks) but not females. Both male and female hearts exhibited age-related reductions in ischaemic tolerance, with a significant decline in recovery evident at 16 weeks in males and later, at 20-24 weeks, in females (versus recoveries in hearts at 8 weeks). Strain also modified tolerance to ischaemia, with similar responses in hearts from C57BL/6, 129/sv, Quackenbush Swiss and FVBN mice, but substantially greater tolerance in BALB/c hearts. In terms of vascular function, baseline coronary flow (20-25 ml min(-1) g(-1)) was 50-60% of maximally dilated flows, and coronary reactive and functional hyperaemic responses were pronounced (up to 4-fold elevations in flow in hearts lacking ventricular balloons). These data indicate that attention to age (and sex) of mice will reduce variability in contractile function and ischaemic responses. Even small differences in perfusion fluid [Ca(2+)] also significantly modify tolerance to ischaemia (whereas modest shifts in heart rate do not impact). Ischaemic responses are additionally strain dependent, with BALB/c hearts displaying greatest intrinsic tolerance. Finally, the model is applicable to the study of vascular reactivity, providing large responses and excellent reproducibility.


An item-response theory analysis of DSM-IV alcohol-use disorder criteria and "binge" drinking in undergraduates.
This is the first study to examine the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), criteria for alcohol-use disorders and heavy episodic (or "binge") drinking in a college sample using item-response theory (IRT) analysis. IRT facilitates assessment of the severity of the criteria, their ability to distinguish between those at greatest and lowest risk, and the value of adding a "binge" drinking criterion. In a sample of undergraduate drinkers (n = 353), we conducted factor analyses to determine whether the criteria best fit a one- or two-factor structure. We then conducted IRT analyses to obtain item-characteristic curves indicating the probability of endorsing a criterion at increasing levels of alcohol-use-disorder risk. These analyses were first conducted including current (i.e., past-year) DSM-IV alcohol-use-disorder criteria only and then rerun adding weekly "binge" drinking. A single-factor model of the DSM-IV criteria did not differ significantly from a two-factor model. After including "binge" drinking, two factors fit the data slightly better than one factor but with a dominant single factor. Withdrawal was the most severe criterion, whereas tolerance and "larger/longer" were the least severe. Time spent drinking and a combined social/legal difficulties criterion had the best ability to discriminate those at greatest and lowest risk for an alcohol-use disorder. "Binge" drinking showed both low discrimination and low severity. To our knowledge, this is the first study to examine DSM-IV criteria in an undergraduate sample using IRT. In this sample, abuse and dependence were intermixed on a continuum of severity, and "binge" drinking was in the least severe region.


Long-chain n-alkane biodegradation coupling to methane production in an enriched culture from production water of a high-temperature oil reservoir.
Paraffinic n-alkanes (C22-C30), crucial portions of residual oil, are generally considered to be difficult to be biodegraded owing to their general solidity at ambient temperatures and low water solubility, rendering relatively little known about metabolic processes in different methanogenic hydrocarbon-contaminated environments. Here, we established a methanogenic C22-C30 n-alkane-degrading enrichment culture derived from a high-temperature oil reservoir production water. During two-year incubation (736 days), unexpectedly significant methane production was observed. The measured maximum methane yield rate (164.40 μmol L-1 d-1) occurred during the incubation period from day 351 to 513. The nearly complete consumption (> 97%) of paraffinic n-alkanes and the detection of dicarboxylic acids in n-alkane-amended cultures indicated the biotransformation of paraffin to methane under anoxic condition. 16S rRNA gene analysis suggested that the dominant methanogen in n-alkane-degrading cultures shifted from Methanothermobacter on day 322 to Thermoplasmatales on day 736. Bacterial community analysis based on high-throughput sequencing revealed that members of Proteobacteria and Firmicutes exhibiting predominant in control cultures, while microorganisms affiliated with Actinobacteria turned into the most dominant phylum in n-alkane-dependent cultures. Additionally, the relative abundance of mcrA gene based on genomic DNA significantly increased over the incubation time, suggesting an important role of methanogens in these consortia. This work extends our understanding of methanogenic paraffinic n-alkanes conversion and has biotechnological implications for microbial enhanced recovery of residual hydrocarbons and effective bioremediation of hydrocarbon-containing biospheres.


Presence of extensive Wolbachia symbiont insertions discovered in the genome of its host Glossina morsitans morsitans.
Tsetse flies (Glossina spp.) are the cyclical vectors of Trypanosoma spp., which are unicellular parasites responsible for multiple diseases, including nagana in livestock and sleeping sickness in humans in Africa. Glossina species, including Glossina morsitans morsitans (Gmm), for which the Whole Genome Sequence (WGS) is now available, have established symbiotic associations with three endosymbionts: Wigglesworthia glossinidia, Sodalis glossinidius and Wolbachia pipientis (Wolbachia). The presence of Wolbachia in both natural and laboratory populations of Glossina species, including the presence of horizontal gene transfer (HGT) events in a laboratory colony of Gmm, has already been shown. We herein report on the draft genome sequence of the cytoplasmic Wolbachia endosymbiont (cytWol) associated with Gmm. By in silico and molecular and cytogenetic analysis, we discovered and validated the presence of multiple insertions of Wolbachia (chrWol) in the host Gmm genome. We identified at least two large insertions of chrWol, 527,507 and 484,123 bp in size, from Gmm WGS data. Southern hybridizations confirmed the presence of Wolbachia insertions in Gmm genome, and FISH revealed multiple insertions located on the two sex chromosomes (X and Y), as well as on the supernumerary B-chromosomes. We compare the chrWol insertions to the cytWol draft genome in an attempt to clarify the evolutionary history of the HGT events. We discuss our findings in light of the evolution of Wolbachia infections in the tsetse fly and their potential impacts on the control of tsetse populations and trypanosomiasis.


People and organizational issues in research systems implementation.
Knowledge about people and organizational issues pertinent to implementation and maintenance of clinical systems has grown steadily over the past fifteen years. Less is known about implementation of systems used for clinical and biomedical research. In conjunction with current National Institutes of Health Roadmap efforts that promote translational research, these issues should now be identified and addressed. During the 2007 American College of Medical Informatics Symposium, members discussed behavioral aspects of translational informatics. This article summarizes that discussion, which covered organizational issues, implications of how knowledge about clinical systems implementation can inform research systems implementation, and those issues unique to each kind of system.


Molecular causes of the aberrant choline phospholipid metabolism in breast cancer.
Proton magnetic resonance spectroscopy ((1)H MRS) consistently detects significant differences in choline phospholipid metabolites of malignant versus benign breast lesions. It is critically important to understand the molecular causes underlying these metabolic differences, because this may identify novel targets for attack in cancer cells. In this study, differences in choline membrane metabolism were characterized in breast cancer cells and normal human mammary epithelial cells (HMECs) labeled with [1,2-(13)C]choline, using (1)H and (13)C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-(13)C]choline for long and short periods of time to distinguish between catabolic and anabolic pathways in choline metabolism. Gene expression analysis using microarrays was performed to understand the molecular mechanisms underlying these changes. Breast cancer cells exhibited increased phosphocholine (PC; P < 0.001), total choline-containing metabolites (P < 0.01), and significantly decreased glycerophosphocholine (P < 0.05) compared with normal HMECs. Decreased (13)C-enrichment was detected in choline (P < 0.001) and phosphocholine (P < 0.05, P < 0.001) of breast cancer cells compared with HMECs, indicating a higher metabolic flux from membrane phosphatidylcholine to choline and phosphocholine in breast cancer cells. Choline kinase and phospholipase C were significantly overexpressed, and lysophospholipase 1, phospholipase A2, and phospholipase D were significantly underexpressed, in breast cancer cells compared with HMECs. The magnetic resonance spectroscopy data indicated that elevated phosphocholine in breast cancer cells was primarily attributable to increased choline kinase activity and increased catabolism mediated by increased phospholipase C activity. These observations were consistent with the overexpression of choline kinase and phospholipase C detected in the microarray analyses.


The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway.
The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.


[Ceruloplasmin in the diagnosis of gynecologic tumors].
Serum caeruloplasmin levels were measured in women with ovarian, collum and corpus carcinomata using three different assay methods. The enzyme assay gave a median value of 0.26 g/l, the SPALT (solid phase antigen luminescence technique) a median value of 0.78 g/l and the ILMA (immunoluminometric assay) a median of 0.44 g/l. A group of blood donors, taken as the reference group, had a median value of 0.29 g/l in the SPALT assay, which was significantly lower than in the tumour group measured by this method. The ILMA and enzymatic assay values were not significantly different from those measured in the reference group. The differences in the SPALT method can be ascribed to the possibility that this assay measures not only "intact" caeruloplasmin but also paraneoplastic substances with "caeruloplasmin-like immunoactivity". The median of the concentration of tissue polypeptide antigen (TPA) was significantly different from those measured in the reference group. The concentrations of carcinoembryonic antigen (CEA) were not different from the reference group.


The effect of octreotide on the prevention of acute pancreatitis and hyperamylasemia after diagnostic and therapeutic ERCP.
Acute pancreatitis is a serious complication of diagnostic and therapeutic Endoscopic Retrograde Cholangiopancreatography (ERCP). In addition, serum pancreatic enzymes increase without symptoms in about 40-50% of patients undergoing these endoscopic procedures. In order to evaluate the efficacy of octreotide in the prevention of these complications, we performed this randomised, prospective study. We studied 73 patients (31 males, 42 females), mean age 63.3 +/- 12.9 years (range 28-87 yrs). The patients were randomly allocated into two groups (A and B). Group A (37 patients) was given 0.1 mg of octreotide subcutaneously 30 min before and 8 and 16 hours after the procedure, and group B (36 patients) was given a placebo. Serum amylase was measured 30 min before and 3 and 6 hrs after ERCP. All patients were subjected to ultrasonography for signs of pancreatic inflammation. There was no statistically significant difference between the two groups concerning age, sex and indication for ERCP. Endoscopic sphincterotomy (ES) was performed in 14 patients of group A and 10 patients of group B. There were 4 cases of acute pancreatitis in each group and the mean serum amylase at 3 and 6 hrs was comparable (494/676 and 429/582 IU/L, respectively). In comparing patients who were subjected to either diagnostic or therapeutic ERCP, there was no statistically significant difference concerning episodes of acute pancreatitis and the level of serum amylase. Octreotide does not seem to prevent acute pancreatitis and hyperamylasaemia after diagnostic and therapeutic ERCP.


Phase variation: evolution of a controlling element.
Phase variation in bacteria is regulated by homologous recombination at a specific DNA site. This recombinational event causes the inversion of a 970-base-pair DNA sequence that includes the promoter necessary for transcription of a flagellar gene. The invertible segment is flanked by two sites that are necessary for the inversion and contains a gene (hin) whose product mediates the inversion event. The hin gene shows extensive homology with the TnpR gene carried on the Tn3 transposon. It is also homologous with the gin gene carried on bacteriophage mu. These relationships suggest that the phase variation system may have evolved by the association of a transposon with a resident gene and the subsequent specialization of these elements to regulate flagellar antigen expression.


A Case of Proliferative Sarcoid Retinopathy with Sarcoid Nodules in Tissue Obtained During Vitrectomy.
Background: In previous studies, intraocular proliferative tissues obtained from proliferative sarcoid retinopathy cases during vitrectomy have been examined histopathologically. However, there is no report of identification of sarcoid nodules in examined tissues. We performed vitrectomy for a case of proliferative sarcoid retinopathy with extensive proliferative changes. Histopathologically, sarcoid nodules were identified in the fibrovascular membranes.Case: A 25-year-old man was treated for sarcoid uveitis in the right eye by his local ophthalmologist. Lens aspiration was performed for complicated cataract. He was referred to our hospital with vitreous opacity and traction retinal detachment which occurred after the surgery.Findings: The vitreous opacity was so severe that details of the fundus were not visible. Traction retinal detachment was suspected from findings of B-mode echography. Vitrectomy was performed, and total retinal detachment due to contraction of the fibrovascular membrane around the optic disc and posterior pole was observed. Sarcoid nodules were identified histopathologically in fibrovascular membranes obtained during vitrectomy.Conclusion: In proliferative sarcoid retinopathy cases, sarcoid lesions may be one of the causes of fibrovascular membrane formation.


Contrast invariance of functional maps in cat primary visual cortex.
Neurons in cat primary visual cortex (V1) are clustered according to their preference for stimulus position, orientation, spatial frequency, and eye of presentation, thereby giving rise to functional maps. It is not known, however, whether a similar arrangement is present for stimulus contrast. Neurons in cat V1 vary considerably in their contrast responses, and might be clustered in a systematic fashion in this respect. Additionally, stimulus contrast might affect other functional maps. For example, there has been debate over whether the contrast threshold of neurons in cytochrome oxidase blobs is lower than elsewhere. Here we have imaged intrinsic signals to measure orientation maps in cat V1 at a range of contrast levels. We determined, on a pixel-by-pixel basis, contrast-response functions and orientation tuning curves. The fit parameters describing contrast responses were more or less uniform: We found no regions where neurons have lower contrast threshold than elsewhere. If such regions do exist, their functional maps must be substantially weaker than maps of orientation preference. Moreover, we found that contrast has no impact on maps of orientation preference: The orientation selectivity of each pixel is invariant with stimulus contrast. The contrast invariance that we demonstrate at the level of maps is well known at the level of single neurons. It suggests that neurons contributing to a pixel response generally have similar orientation preferences or similar contrast responses. The latter explanation is likely to hold in pinwheel centers, where preferred orientation of nearby neurons can differ markedly. In summary, our data suggest that contrast is represented uniformly over the surface of cat V1, and changes in contrast do not affect maps of orientation preference.


Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies.
Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B. One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A. This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin. The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide. Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components. The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C. difficile by immunoaffinity chromatography. The second antibody, the G-2 MAb, cross-reacted with toxins A and B. The cross-reaction was confirmed by immunoblot analysis. These results show that toxins A and B share an epitope and suggest that they have a common subunit. The G-2 MAb did not neutralize or precipitate either toxin. The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.


Tenascin: a potential role in human arteriovenous PTFE graft failure.
To investigate the involvement of tenascin (TN) in human neointimal hyperplastic lesion formation, we studied 12 human arteriovenous (AV) polytetrafluoroethylene (PTFE) loop grafts removed at the time of graft revision. Immunoperoxidase technique was used to determine TN and proliferating cell nuclear antigen (PCNA) as an index of cell proliferation. The venous anastomotic neointimal hyperplastic lesion was analyzed as three layers, each one-third of the thickness. TN was distributed in the neointima as follows: (1) in the luminal layer, all lesions were intensely stained; (2) in the middle layer, 9 of 12 (75%) lesions had moderate reactivity; (3) in the deep layer near the PTFE grafts, 3 of 12 (25%) lesions were strongly stained and 5 of 12 (42%) had moderate reactivity; and (4) 90 +/- 6% of microvessels within the neointima showed intense periendothelial staining. PCNA indices were as follows: luminal layer, 29 +/- 11%; middle layer, 9 +/- 5%; deep layer near the graft, 9 +/- 7%; and microvessel containing intimal fields, 67 +/- 4%. The cell proliferation rate was significantly higher in the luminal layer than in the middle or deep layers of neointima (P < 0.05). The PCNA index in microvessel-containing intimal fields was three to eight times that of avascular fields (P < 0.001). These data demonstrate that TN is distributed in a pattern similar to that of cell proliferation in human neointimal hyperplastic lesions. The results suggest that TN expression may have a potential role in neovascularization and neointimal growth in human AV PTFE graft failure.


The role of high-frequency audiometry in early detection of ototoxicity.
Ototoxicity is one of the unwanted side-effects of a number of medical drugs. As ototoxicity appears to be most pronounced in the higher frequencies, it can be assessed at an earlier stage by using high-frequency audiometry from 8 to 20 kHz. We have investigated the precision of these measurements. In spite of inaccuracies of headphone positioning, which may influence especially the high-frequency thresholds, standard deviations for frequencies above 8 kHz were obtained that were nearly as small as for audiometry up to 8 kHz. Age-related reference curves for the higher frequencies are presented. The results on 100 ears of patients treated with platinum-derivatives show the importance of high-frequency audiometry for the early detection of ototoxicity.


Detection of dose delivery variations on TomoTherapy using on-board detector based verification.
A clinical case of delivery dose deviations on a TomoTherapy treatment was discovered during a patient specific treatment quality assurance (QA) verification. An in-house developed QA system, MCLogQA, for TomoTherapy has been implemented in our clinic for patient specific treatment QA. The MCLogQA system utilizes the log file and detector-based multileaf collimator (MLC) leaf opening time (LOT) to assess accuracy of treatment plan delivery. Recently, the MCLogQA system discovered >10% dose deviation for a low dose/fraction treatment plan. To verify the adequacy of the MCLogQA result, a delivery quality assurance (DQA) plan was created and performed. The treatment plan was also transferred to a second TomoTherapy unit and planning system to investigate if the plan-delivery deviation was unit dependent. Further testing was carried out in phantom plans. MCLogQA showed MLC LOT was on average 2.4% higher than the planned LOT, resulting in 3.5% increase in mean dose, and 14% increase in dose to 1 cc volume of max dose in PTV. Independent DQA verification confirmed the MCLogQA result. For the transferred treatment plan delivery, the MCLogQA also showed an average increase of 6.6% in MLC LOT, resulting in increases in mean dose by 9.3% and dose to 1 cc volume of max dose in PTV by 16%. The inaccurate MLC LOT was a result of a poor latency model at very small LOT. Phantom testing confirmed low LOT will result in relatively large dosimetric variation, and detector-based MCLogQA will detect differences in planned and measured LOT. Accuracy in TomoTherapy treatment delivery can be susceptible to LOT uncertainty. Using MCLogQA for QA verification not only validates the treatment delivery, but also provides information on LOT variation and comprehensive dose distribution. This information can help decision making when large plan-delivery deviation occurs.


Nomograms for prediction of long-term survival in elderly patients after partial hepatectomy for hepatocellular carcinoma.
Partial hepatectomy is an important treatment for elderly patients with hepatocellular carcinoma. However, prediction of long-term outcomes of an individual elderly patient after partial hepatectomy still is lacking. This study aimed to develop 2 nomograms to pre- or postoperatively predict overall survival for these patients. Of the 528 elderly patients (aged ≥65 years) who underwent partial hepatectomy for hepatocellular carcinoma at the Eastern Hepatobiliary Surgery Hospital between 2008 and 2011, 425 patients served as a training cohort to develop pre- and postoperative nomograms, and the remaining 103 patients comprised a validation cohort. The Cox proportional hazards model was used for univariate and multivariable analyses of tumor recurrence and overall survival. Discrimination and calibration of the models were measured using the concordance index, calibration plots, and Kaplan-Meier curves. Based on preoperative data, the independent risk factors of overall survival were age ≥75 years, Charlson score, α-fetoprotein ≥20 μg/L, hepatitis B virus-deoxyribonucleic acid ≥104 IU/mL, and tumor diameter. Based on postoperative data, nonanatomic hepatectomy, absence of tumor encapsulation, and presence of microvascular invasion were additional independent risk factors. These independent predictors were incorporated into the pre- and postoperative nomograms, respectively. The concordance indexes of the 2 nomograms for overall survival prediction were 0.70 (95% confidence interval, 0.67-0.74) and 0.72 (0.69-0.78), respectively. Both nomograms accurately predicted 1-, 3-, and 5-year overall survival probability, and their predictive performances were optimally validated. The proposed 2 nomograms showed good individualized predictive performance in elderly patients with hepatocellular carcinoma before and after partial hepatectomy.


Implications of attachment theory and research for the assessment and treatment of eating disorders.
In this paper, we review the research literature on attachment and eating disorders and suggest a framework for assessing and treating attachment functioning in patients with an eating disorder. Treatment outcomes for individuals with eating disorders tend to be moderate. Those with attachment-associated insecurities are likely to be the least to benefit from current symptom-focused therapies. We describe the common attachment categories (secure, avoidant, anxious), and then describe domains of attachment functioning within each category: affect regulation, interpersonal style, coherence of mind, and reflective functioning. We also note the impact of disorganized mental states related to loss or trauma. Assessing these domains of attachment functioning can guide focused interventions in the psychotherapy of eating disorders. Case examples are presented to illustrate assessment, case formulation, and group psychotherapy of eating disorders that are informed by attachment theory. Tailoring treatments to improve attachment functioning for patients with an eating disorder will likely result in better outcomes for those suffering from these particularly burdensome disorders.


Evaluation of intraoperative magnetic resonance imaging/ultrasound fusion optimization for low-dose-rate prostate brachytherapy.
Intraoperative planning with transrectal ultrasound (US) is used for accurate seed placement and optimal dosimetry in prostate brachytherapy. However, prostate magnetic resonance imaging (MRI) has shown superiority in delineation of prostate anatomy. Accordingly, MRI/US fusion may be useful for accurate intraoperative planning. We analyzed planning with MRI/US fusion to compare differences in dosimetry and volume to that derived from the postoperative computed tomography (CT). Twenty patients underwent preoperative prostate MRI, which was fused intraoperatively with US during prostate brachytherapy. Intraoperative 125I or 103Pd seed placement was modified by the use of MRI fusion when indicated. Following implantation, dose comparisons were made between data derived from MRI/US and that from post-operative CT scans. Plan parameters analyzed included the D90 (dose to 90% of the prostate), rectal D30, V30 (volume of the rectum receiving 30 percent of dose), and prostate V100. The median number of seeds implanted per patient was seventy-six. The MRI measured prostate volume, which was on average 4.47 cc larger than the CT measured prostate volume. In 9 patients, the apex of the prostate was better identified under MRI with the fusion protocol, and an average of 4 fewer seeds were required to be placed in the apex/urinary sphincter region. Both MRI and US individually showed a reduced intraoperative prostate D90 in comparison to the postoperative CT, with a larger mean difference for MRI in comparison with US (9.71 vs. 4.31 Gy, p = 0.007). This was also true for the prostate V100 (5.18 vs. 2.73 cc, p = 0.009). Post-operative CT underestimated rectal D30 and V30 in comparison to both MRI and US with MRI showing a larger mean difference than US for D30 (40.64 vs. 35.92 Gy, p = 0.04) and V30 (50.20 vs. 44.38 cc, p = 0.009). The MRI/US fusion demonstrated greater prostate volume compared to standard CT/US based planning likely due to the better resolution of the prostate apex. Furthermore, rectal dose was underestimated with CT vs. MRI based planning. Additional study is required to assess long-term clinical implications of disease control and effects on long-term toxicity, especially as related to the rectum and urinary sphincter. MRI/US intraoperative fusion may improve prostate dosimetry while sparing the rectum and urethra, potentially impacting disease control and late toxicity.


Novel Phakopsora pachyrhizi extracellular proteins are ideal targets for immunological diagnostic assays.
Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots.


Predicting Dinitrogen Activation via Transition-Metal-Involved [4+2] Cycloaddition Reaction.
As the strongest triple bond in nature, the N≡N triple bond activation has always been a challenging project in chemistry. On the other hand, since the award of the Nobel Prize in Chemistry in 1950, the Diels-Alder reaction has served as a powerful and widely applied tool in the synthesis of natural products and new materials. However, the application of the Diels-Alder reaction to dinitrogen activation remains less developed. Here we first demonstrate that a transition-metal-involved [4+2] Diels-Alder cycloaddition reaction could be used to activate dinitrogen without an additional reductant by density functional theory calculations. Further study reveals that such a dinitrogen activation by 1-metalla-1,3-dienes screened out from a series of transition metal complexes (38 species) according to the effects of metal center, ligand, and substituents can become favorable both thermodynamically (with an exergonicity of 28.2 kcal mol-1 ) and kinetically (with an activation energy as low as 13.8 kcal mol-1 ). Our findings highlight an important application of the Diels-Alder reaction in dinitrogen activation, inviting experimental chemists' verification.


Mutation characterization of CFTR gene in 206 Northern Irish CF families: thirty mutations, including two novel, account for approximately 94% of CF chromosomes.
A variety of mutation detection techniques, including restriction endonuclease digestion, allele specific oligonucleotides, and automated fluorescent sequencing, were used in the identification of 15 CFTR mutations representing 86.7% of CF chromosomes in 206 Northern Irish cystic fibrosis (CF) families. A systematic analysis of the 27 exons and intron/exon boundaries of the CFTR gene was performed using denaturing gradient gel electrophoresis (DGGE) in an attempt to characterise the 55 unknown CF mutations in 51 patients. Twenty different mutations were detected by DGGE on 30 chromosomes accounting for a further 7.3% of CF alleles. Fifteen of these mutations had not previously been found in Northern Ireland, and two are novel, M1I(G > T) and V562L. In total, 30 CFTR mutations account for 93.9% of the 412 Northern Irish CF chromosomes tested. The three major CF mutations in Northern Ireland are delta F508, G551D, and R117H with respective frequencies of 68.0%, 5.1%, and 4.1%. The efficacy of the DGGE technique was proven by the detection of 77 out of 77 control variants from all the CFTR exons. DGGE is a highly efficient and sensitive method for mutation screening especially in large genes where the mutation spectrum is known to be heterogeneous.


Two trypanocidal dipeptides from the roots of Zapoteca portoricensis (Fabaceae).
Zapoteca portoricensis (Jacq) HM Hernández is used with remarkable efficacy in ethnomedicinal management of tonsillitis in the Eastern part of Nigeria. Previous pharmacological studies have validated the antiinflammatory and antimicrobial activities of the crude extract. In this study, two dipeptides, saropeptate (aurantiamide acetate) and anabellamide, were isolated from the methanol root extract of Zapoteca portoricensis and their chemical structures deduced by one dimensional and two dimensional NMR and mass spectrometry. These compounds were isolated for the first time from this plant, and no report has been found on their previous isolation from the genus Zapoteca. Evaluation of their trypanocidal activity showed that compound 1 exhibited potent activity against Trypanosoma brucei rhodesiense with an IC50 value of 3.63 μM and selectivity index of 25.3.


In tribute to Bob Blanchard: Divergent behavioral phenotypes of 16p11.2 deletion mice reared in same-genotype versus mixed-genotype cages.
Mouse models offer indispensable heuristic tools for studying genetic and environmental causes of neuropsychiatric disorders, including autism. Development of useful animal models of complex human behaviors depends not only on extensive knowledge of the human disease, but also on a deep understanding of animal behavior and ethology. Robert and Caroline Blanchard pioneered a number of elegant social paradigms in rodents. Their early work led to systematic delineations of rodent naturalist defensive behaviors,which were proven to be highly useful models of human psychiatric disorders, including fear and anxiety. Their work using the Visible Burrow System to study social stress in rats represented an unprecedented approach to study biological mechanisms of depression. In recent years, their extensive knowledge of mouse behavior and ethology enabled them to quickly become leading figures in the field of behavioral genetics of autism. To commemorate Robert Blanchard's influences on animal models of human psychiatric disorders, here we describe a study conceptualized and led by Mu Yang who was trained as a graduate student in the Blanchard laboratory in the early 2000s. This investigation focuses on social housing in a genetic mouse model of 16p11.2 deletion syndrome. Heterozygous deletions and duplications of a segment containing about 29 genes on human chromosome 16 appear in approximately 0.5–1% of all cases of autism. 16p11.2 deletion syndrome is also associated with intellectual disabilities and speech impairments. Our previous studies showed that a mouse model of 16p11.2 deletion syndrome exhibited deficits in vocalizations and novel object recognition, as compared to wildtype littermate control cagemates. In the spirit of Bob Blanchard's careful attention to the role of social dominance in rodent behaviors, we became interested in the question of whether behavioral outcomes of a mutation differ when mutants are housed in mixed genotype cages, versus housing only mutants together in one group cage, and only wildtype littermates together in another group cage after weaning. 16p11.2 deletion presented a particularly good model organism to investigate this question, because the heterozygotes are smaller than their wildtype littermates, and may therefore become subordinate to their larger cagemates.Wildtype and heterozygotes were housed with cagemates of the same genotype (same-genotype cage) or with cagemates of the opposite genotype (mixed-genotype cage). Current results replicated social vocalization and object recognition deficits that we previously found in heterozygotes living in mixed-genotype cages. In contrast, heterozygotes that lived in same-genotype cages emitted normal numbers of vocalizations during male–female interactions, and displayed normal novel object recognition, indicating that the deletion per se was not sufficient to cause cognitive or social deficits. Social approach, same-sex social interaction, anxiety-related behavior, depression-related behavior, and open field exploration were not different between genotypes, and were not affected by housing in mixed versus in same-genotype cages. These findings suggest that elements of the home cage social environment could interact with genotype to impact aspects of disease phenotypes. Current findings are discussed as potentially reflecting behavioral deficits resulted from social stress, as inspired by a seminal paper by Bob and Caroline Blanchard [1].


teashirt is required for head-versus-tail regeneration polarity in planarians.
Regeneration requires that the identities of new cells are properly specified to replace missing tissues. The Wnt signaling pathway serves a central role in specifying posterior cell fates during planarian regeneration. We identified a gene encoding a homolog of the Teashirt family of zinc-finger proteins in the planarian Schmidtea mediterranea to be a target of Wnt signaling in intact animals and at posterior-facing wounds. Inhibition of Smed-teashirt (teashirt) by RNA interference (RNAi) resulted in the regeneration of heads in place of tails, a phenotype previously observed with RNAi of the Wnt pathway genes β-catenin-1, wnt1, Dvl-1/2 or wntless. teashirt was required for β-catenin-1-dependent activation of posterior genes during regeneration. These findings identify teashirt as a transcriptional target of Wnt signaling required for Wnt-mediated specification of posterior blastemas.


The fate of pharmaceutical chemicals in the aquatic environment.
Increased demands for potable water, especially where supplies are drawn from lowland rivers has necessitated a greater degree of water re-use. As water undertakings have a duty to maintain the wholesome quality of potable water supplies, increasing concern is being expressed over the presence of organic micro-contaminants (contaminants found at microgram litre-1 concentrations). This study outlines some of the problems encountered in assessing the risk from pharmaceutical chemicals which might enter the water cycle from domestic and industrial sources. Analytical chemistry was of value for only a few of the 200 compounds studied. However, much useful information was derived from the human metabolic routes of the drugs and is collated in Appendix I. Biodegradation studies and other ecotoxicity/environmental toxicology data may be required to a greater extent in the future. Particular consideration is given to vulnerable sections of the population.


Making Lemonade Together -- How Program Directors, Applicants, and Medical Schools Can Thrive During the Upcoming Interview Season.
This article was migrated. The article was marked as recommended. The virtual residency interview process ushers in a new era of medical education. Many stakeholders are increasingly concerned as validated recommendations regarding Match success appear less reliable, fossilized rules have become increasingly fluid, and traditional streams of communication have become inadequate. Program directors will look to sell their program using unvalidated methods. Applicants will make life-altering decisions using fewer data points than historically available. Medical schools will endeavor to advise their students as they gear up for breaking new ground. In this piece, we introduce considerations and recommendations for the main players involved in the virtual interview process. If each party prioritizes teamwork and communication, we can collectively tackle the challenges of the upcoming cycle and turn lemons into lemonade.


The Impact of Postoperative Intensive Care Unit Admission on Postoperative Hospital Length of Stay and Costs: A Prespecified Propensity-Matched Cohort Study.
In this prespecified cohort study, we investigated the influence of postoperative admission to the intensive care unit versus surgical ward on health care utilization among patients undergoing intermediate-risk surgery. Of adult surgical patients who underwent general anesthesia without an absolute indication for postoperative intensive care unit admission, 3530 patients admitted postoperatively to an intensive care unit were matched to 3530 patients admitted postoperatively to a surgical ward using a propensity score based on 23 important preoperative and intraoperative predictor variables. Postoperative hospital length of stay and hospital costs were defined as primary and secondary end points, respectively. Among patients with low propensity for postoperative intensive care unit admission, initial triage to an intensive care unit was associated with increased postoperative length of stay (incidence rate ratio, 1.69 [95% CI, 1.59-1.79]; P < .001) and hospital costs (incidence rate ratio, 1.92 [95% CI, 1.81-2.03]; P < .001). By contrast, postoperative intensive care unit admission of patients with high propensity was associated with decreased postoperative length of stay (incidence rate ratio, 0.90 [95% CI, 0.85-0.95]; P < .001) and costs (incidence rate ratio, 0.92 [95% CI, 0.88-0.97]; P = .001). Decisions regarding postoperative intensive care unit resource utilization were influenced by individual preferences of anesthesiologists and surgeons. In patients with an unclear indication for postoperative critical care, intensive care unit admission may negatively impact postoperative hospital length of stay and costs. Postoperative discharge disposition varies substantially based on anesthesia and surgical provider preferences but should optimally be driven by an objective assessment of a patient's status at the end of surgery.


Enhanced Water Barrier Properties of Surfactant-Free Polymer Films Obtained by MacroRAFT-Mediated Emulsion Polymerization.
The presence of low-molar-mass surfactants in latex films results in detrimental effects on their water permeability, gloss, and adhesion. For applications such as coatings, there is a need to develop formulations that do not contain surfactants and have better water barrier properties. Having previously reported the synthesis of surfactant-free latex particles in water using low amounts (<2 wt %) of chains synthesized by controlled radical polymerization (Lesage de la Haye et al. Macromolecules 2017, 50, 9315-9328), here we study the water barrier properties of films made from these particles and their application in anticorrosion coatings. When films cast from aqueous dispersions of acrylate copolymer particles stabilized with poly(sodium 4-styrenesulfonate) (PSSNa) were immersed in water for 3 days, they sorbed only 4 wt % water. This uptake is only slightly higher than the value predicted for the pure copolymer, indicating that the negative effects of any particle boundaries and hydrophilic-stabilizing molecules are minimal. This sorption of liquid water is 5 times lower than what is found in films cast from particles stabilized with the same proportion of poly(methacrylic acid) (PMAA), which is more hydrophilic than PSSNa. In water vapor with 90% relative humidity, the PSSNa-based film had an equilibrium sorption of only 4 wt %. A small increase in the PMAA content has a strong and negative impact on the barrier properties. Nuclear magnetic resonance relaxometry on polymer films after immersion in water shows that water clusters have the smallest size in the films containing PSSNa. Furthermore, these films retain their optical clarity during immersion in liquid water for up to 90 min, whereas all other compositions quickly develop opacity ("water whitening") as a result of light scattering from sorbed water. This implies a remarkably complete coalescence and a very small density of defects, which yields properties matching those of some solvent-borne films. The latex stabilized with PSSNa is implemented as the binder in a paint formulation for application as an anticorrosive barrier coating on steel substrates and evaluated in accelerated weathering and corrosion tests. Our results demonstrate the potential of self-stabilized latex particles for the development of different applications, such as waterborne protective coatings and pressure-sensitive adhesives.


Large-Cavity Coronoids with Different Inner and Outer Edge Structures.
Coronoids, polycyclic aromatic hydrocarbons with geometrically defined cavities, are promising model structures of porous graphene. Here, we report the on-surface synthesis of C168 and C140 coronoids, referred to as [6]- and [5]coronoid, respectively, using 5,9-dibromo-14-phenylbenzo[m]tetraphene as the precursor. These coronoids entail large cavities (>1 nm) with inner zigzag edges, distinct from their outer armchair edges. While [6]coronoid is planar, [5]coronoid is not. Low-temperature scanning tunneling microscopy/spectroscopy and noncontact atomic force microscopy unveil structural and electronic properties in accordance with those obtained from density functional theory calculations. Detailed analysis of ring current effects identifies the rings with the highest aromaticity of these coronoids, whose pattern matches their Clar structure. The pores of the obtained coronoids offer intriguing possibilities of further functionalization toward advanced host-guest applications.


Low prevalence of influenza A strains with resistance markers in Brazil during 2017-2019 seasons.
The influenza A virus (IAV) is of a major public health concern as it causes annual epidemics and has the potential to cause pandemics. At present, the neuraminidase inhibitors (NAIs) are the most widely used anti-influenza drugs, but, more recently, the drug baloxavir marboxil (BXM), a polymerase inhibitor, has also been licensed in some countries. Mutations in the viral genes that encode the antiviral targets can lead to treatment resistance. Worldwide, a low prevalence of antiviral resistant strains has been reported. Despite that, this situation can change rapidly, and resistant strain surveillance is a priority. Thus, the aim of this was to evaluate Brazilian IAVs antiviral resistance from 2017 to 2019 through the identification of viral mutations associated with reduced inhibition of the drugs and by testing the susceptibility of IAV isolates to oseltamivir (OST), the most widely used NAI drug in the country. Initially, we analyzed 282 influenza A(H1N1)pdm09 and 455 A(H3N2) genetic sequences available on GISAID. The amino acid substitution (AAS) NA:S247N was detected in one A(H1N1)pdm09 strain. We also identified NA:I222V (n = 6) and NA:N329K (n = 1) in A(H3N2) strains. In addition, we performed a molecular screening for NA:H275Y in 437 A(H1N1)pdm09 samples, by pyrosequencing, which revealed a single virus harboring this mutation. Furthermore, the determination of OST IC50 values for 222 A(H1N1)pdm09 and 83 A(H3N2) isolates revealed that all isolates presented a normal susceptibility profile to the drug. Interestingly, we detected one A(H3N2) virus presenting with PA:E119D AAS. Moreover, the majority of the IAV sequences had the M2:S31N adamantanes resistant marker. In conclusion, we show a low prevalence of Brazilian IAV strains with NAI resistance markers, in accordance with what is reported worldwide, indicating that NAIs still remain an option for the treatment of influenza infections in Brazil. However, surveillance of influenza resistance should be strengthened in the country for improving the representativeness of investigated viruses and the robustness of the analysis.


How to Approach Difficult Cases of AVNRT.
Our approach to the ablation of atrioventricular nodal reciprocating tachycardia (AVNRT), the most common supraventricular tachycardia, is as follows: We first attempt ablation in the right atrial posteroseptum anterior to the coronary sinus ostium with a 4-mm non-irrigated tip catheter. If ablation within the triangle of Koch is unsuccessful with radiofrequency (RF), we switch to cryoablation and target a more superior (mid septal) region. We also utilize cryoablation if RF ablation produces transient VA block (absence of retrograde conduction during junctional rhythm) or a fast junctional rhythm (<350 msec). If cryoablation were to fail, or is not available, we would then suggest ablation within the coronary sinus targeting the roof (2-4 cm from the os) using a 3.5-mm irrigated tip catheter. If tachycardia were still inducible despite these measures, we would then proceed with transseptal puncture (given our greater experience with this over a retrograde aortic approach) and perform RF ablation along the posteroseptal left atrium and inferoseptal mitral annulus utilizing an irrigated tip catheter. In our experience, cryoablation reliably results in elimination of the slow pathway. The only left atrial ablation for AVNRT at our institution in the past year was performed because a patent foramen ovale allowed for rapid left atrial access, facilitating left atrial ablation of the slow pathway.


Long-Term Treatment of Unresectable Pseudomyxoma Peritonei with Multiple Treatments of Intratumoural Bromelain and Acetylcysteine (BromAc®): A Case Report.
Pseudomyxoma peritonei is a rare peritoneal malignancy characterized by the progressive accumulation of mucinous material and tumour within the abdomen and pelvis. Percutaneous drainage of mucin may be a non-surgical option for relief of symptoms; however, it remains difficult due to the high viscosity of mucin, with numerous case reports reporting difficulty removing material through medium-bore catheters alone. BromAc is a therapy currently undergoing development which dissolves mucinous tumour masses and allows for extraction. This report describes the case of a patient who has had multiple treatments with BromAc over 4 years.


SigWin-detector: a Grid-enabled workflow for discovering enriched windows of genomic features related to DNA sequences.
Chromosome location is often used as a scaffold to organize genomic information in both the living cell and molecular biological research. Thus, ever-increasing amounts of data about genomic features are stored in public databases and can be readily visualized by genome browsers. To perform in silico experimentation conveniently with this genomics data, biologists need tools to process and compare datasets routinely and explore the obtained results interactively. The complexity of such experimentation requires these tools to be based on an e-Science approach, hence generic, modular, and reusable. A virtual laboratory environment with workflows, workflow management systems, and Grid computation are therefore essential. Here we apply an e-Science approach to develop SigWin-detector, a workflow-based tool that can detect significantly enriched windows of (genomic) features in a (DNA) sequence in a fast and reproducible way. For proof-of-principle, we utilize a biological use case to detect regions of increased and decreased gene expression (RIDGEs and anti-RIDGEs) in human transcriptome maps. We improved the original method for RIDGE detection by replacing the costly step of estimation by random sampling with a faster analytical formula for computing the distribution of the null hypothesis being tested and by developing a new algorithm for computing moving medians. SigWin-detector was developed using the WS-VLAM workflow management system and consists of several reusable modules that are linked together in a basic workflow. The configuration of this basic workflow can be adapted to satisfy the requirements of the specific in silico experiment. As we show with the results from analyses in the biological use case on RIDGEs, SigWin-detector is an efficient and reusable Grid-based tool for discovering windows enriched for features of a particular type in any sequence of values. Thus, SigWin-detector provides the proof-of-principle for the modular e-Science based concept of integrative bioinformatics experimentation.


The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles.
Rheumatoid arthritis is a chronic inflammatory disease characterized by destruction of cartilage and bone that is mediated by synovial fibroblasts. To determine the mechanisms by which these cells are activated to produce matrix metalloproteinases (MMPs), the effects of microparticles were investigated. Microparticles are small membrane-bound vesicles whose release from immune cells is increased during activation and apoptosis. Because microparticles occur abundantly in the synovial fluid in rheumatoid arthritis, they could represent novel stimulatory agents. Microparticles derived from T cells and monocytes strongly induced the synthesis of MMP-1, MMP-3, MMP-9, and MMP-13 in fibroblasts. The induction was time-dependent, with effects primarily observed after 36 h; under these conditions, MMP-2, MMP-14, and tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and TIMP-3 were not induced. Microparticles also increased the synthesis of inflammatory mediators including IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), and MCP-2. In Ikappa-B-transfected synovial fibroblasts, MMPs were less inducible by microparticles compared with wild-type fibroblasts. Blocking of TNFalpha and IL-1beta with antibodies against TNFalpha and with IL-1 receptor antagonist did not abrogate stimulation by microparticles. These data provide evidence for a novel mechanism by which vesicles derived from activated or apoptotic immune cells can promote the destructive activity of synovial fibroblasts in rheumatoid arthritis.


Three-dimensional magnetic resonance imaging findings of inner ear anomaly.
Using three-dimensional magnetic resonance imaging (3D-MRI), the membranous labyrinth was examined in normal cases and in a case with bilateral Mondini-type inner ear malformations. Both ears were also examined by high-resolution computed tomography (CT) of the temporal bone. 3D-MRI provided more detail than findings obtained by conventional high-resolution CT and it enabled 3D observations to be made. Detailed examination of maximum intensity projection images along the cochlea axis revealed the cochlear turns in three dimensions.


Improving the gait performance of non-fluid-based swing-phase control mechanisms in transfemoral prostheses.
A prosthetic swing-phase control mechanism simulates the action of leg musculature, aiding gait function by controlling the duration of swing, extent of heel-rise, and by allowing the shank to smoothly decelerate into full knee extension without excessive impact. Non-fluid-based mechanisms have the potential to provide a durable and affordable solution as required in many parts of the world, but the design variables that lead to improved performance of non-fluid-based swing-phase control technologies are not well established. Seven transfemoral amputees were fitted with a prosthetic knee joint and different non-fluid-based swing-phase setups were systematically assessed. Clinical testing included walk tests utilizing a potentiometer (to measure knee flexion angles) and accelerometer (to measure terminal impact decelerations) mounted on the prosthetic limb. As hypothesized, the friction and spring systems improved gait function. This includes an increased walking speed that closely matched high-end hydraulic prosthetic knee joints, decreased and more normal maximum prosthetic knee flexion, decreased flexion duration, and lower terminal impact. Further improvements were obtained using a dual spring system, two springs in series, over the more conventional single spring system. Non-fluid-based swing-phase control mechanisms are simple and significantly improve the performance of prostheses. Their application is ideal where size, weight and cost may be constrained.


Newly developed novel ultrasound technique, defect reperfusion ultrasound imaging, using sonazoid in the management of hepatocellular carcinoma.
The aim of this study is to clarify the usefulness of defect reperfusion ultrasound (US) imaging using Sonazoid in the management of hepatocellular carcinoma (HCC). A total of 33 HCC nodules and 34 local recurring nodules after radiofrequency ablation (RFA), which could not be identified by B-mode US but were depicted by dynamic CT, were studied by defect reperfusion US imaging with Sonazoid. In addition, Kupffer phase Sonazoid-enhanced US in combination with defect reperfusion US imaging were used for screening HCC in 262 consecutive cirrhotic patients. As a result, 33 US undetectable HCC nodules and 34 local recurring HCC nodules were successfully confirmed by Sonazoid-enhanced US with defect reperfusion imaging. Subsequently, RFA was successfully performed in all of 67 HCC nodules with a Sonazoid-enhanced US guidance. A total of 7 small HCCs were depicted and confirmed as HCCs by Kupffer phase surveillance and defect reperfusion US imaging. In conclusion, defect reperfusion US imaging is extremely useful in the depiction and confirmation of US undetectable HCCs as well as in the surveillance of HCC in cirrhotic patients.


BOC-PLAG1, a new fusion gene of pleomorphic adenoma: Identified in a fine-needle aspirate by RNA next-generation sequencing.
Pleomorphic adenoma (PA) is the most common benign salivary gland tumor. Fine-needle aspiration (FNA) of PA exhibits variable combinations of bland ductal epithelial cells, myoepithelial cells, and characteristic magenta fibrillary stroma on Diff-Quik/Romanowsky stain. However, a cellular PA with scant chondromyxoid stroma can be a diagnostic challenge on FNA. Around 70% of PAs have a translocation involving PLAG1 or HMGA2. The presence of either PLAG1 or HMGA2 fusion gene can be used to diagnose PA since they have not been reported in other salivary gland tumors except for carcinoma ex PA. In this case report, we describe a case of cellular PA initially diagnosed on FNA as a "low grade salivary gland neoplasm, favor PA." RNA next-generation sequencing performed on the cell block showed a BOC-PLAG1 fusion gene. The presence of PLAG1 fusion gene in conjunction with cytomorphology supported a diagnosis of PA. The mass was surgically removed and proved to be a cellular PA with scattered foci of chondromyxoid and collagenous stroma. To our knowledge, this is the first reported PA bearing BOC-PLAG1. RNA next-generation sequencing performed on cytology specimens can be helpful in achieving a more specific diagnosis of salivary gland tumors.


Ultrastructure and nuclear architecture of telomeric chromatin revealed by correlative light and electron microscopy.
Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ.


Risk Factors of Central Line-Associated Bloodstream Infection (CLABSI): A Prospective Study From a Paediatric Intensive Care Unit in South India.
Background Central line-associated bloodstream infection (CLABSI) is the most common hospital-acquired infection. However, studies evaluating the factors associated with the risk of CLABSI in pediatric intensive care units (PICU) were limited in India. Objective The objective of our study is to evaluate the association of factors and the etiology causing CLABSI. Study design This is a hospital-based single-center prospective study conducted in the pediatric intensive care unit (PICU) of our tertiary care hospital spanning one year. Participants Children aged between two months to 15 years admitted in the PICU for more than 48 hours with central venous catheterization were included. Pearson's chi-squared test with Yates' continuity correction and logistic regression with odds ratio were calculated by R statistical software (R Foundation for Statistical Computing, Vienna, Austria) and a p-value less than 0.05 was considered statistically significant. Results Our analysis showed that factors such as young age (2-12 months), high pediatric risk of mortality (PRISM III) score (> 15), leukocytosis, neutrophilia, anemia, change of central venous catheter, duration of catheterization (>7 days), exposure to blood products, use of steroids, inotropes, and prophylactic antibiotics were significantly associated with increased risk of CLABSIs with an odds ratio of 4.53, 4.54, 2.91, 4.56, 4.76, 3.74, 2.49, 2.41, 7.22, 6.77 and 5.16 respectively (p<0.05). Further, factors such as older age (>12 months) and low PRISM III score (≤ 15) significantly reduce the risk of CLABSIs by 83.64% and 69.14% respectively (p<0.05). Conclusion In conclusion, our results revealed that factors such as young age, high PRISM III score, leukocytosis, neutrophilia, anemia, change of central venous catheter, duration of catheterization (> 7 days), exposure to blood products during the hospital stay, use of steroids, inotropes, and prophylactic antibiotics were identified as risk factors for CLABSI.


Trends in Dietary Micronutrient Adequacy in Young Adults Over the Latest 25 Years in Japan.
It has been assumed that economically developed countries are well nourished compared to developing countries, but little is known about how economic status affects dietary micronutrient intake in the future childbearing generation. We analyzed the trend of dietary micronutrient adequacy in young adults in Japan, as one of the representative countries with advanced dietary habits and economic progress. We conducted a retrospective analysis using 2 web-accessible databases, namely the Japanese National Health and Nutrition Survey and the World Development Indicators. Japan has been facing a progressive insufficiency of dietary vitamins A and C and iron, especially among young adults, over the past 25 years. The hidden progression of silent malnutrition has become more apparent since the 2010s, coinciding with a series of economic recessions and natural disasters. Given that parental dietary habits play a critical role in ensuring a balanced diet for their children, our findings underscore the importance of proactive nutrition counseling and education, especially for young adults of childbearing age who have been identified as vulnerable to micronutrient deficiencies. In line with this policy, we would like to suggest the use of digital transformation platforms as a potential solution in the future, especially for the digital native population.


Biosynthesis of glucagon in isolated pancreatic islets of guinea pigs.
1. The biosynthesis of glucagon in guinea-pig A(2) cells was investigated by incubation of isolated islets of Langerhans in the presence of [(3)H]tryptophan for periods of up to 14 days. Proteins were extracted from islets and incubation media and analysed by gel filtration. 2. In addition to very-high-molecular-weight (100000) proteins, the principal tryptophan-containing biosynthetic product after incubation for up to 17h was a protein of minimum mol.wt. 9000, which co-eluted on gel filtration with a peak of glucagon-like immunoreactivity, but was apparently devoid of biological activity in a fat-cell assay. A discrete peak of labelled glucagon was only recovered after incubation for at least 6 days. Losses of glucagon during the extraction and rapid secretion of newly synthesized glucagon into incubation media were excluded as reasons for the lack of recovery of labelled hormone from islets after shorter incubations. 3. The 9000-mol.wt. protein was localized to A(2) cells in experiments using B-cell-depleted islets, and to A(2)-cell granules by subcellular fractionation and electron-microscopic radioautography. Only glucagon was secreted into the incubation medium. 4. Possible relationships between the 9000-mol.wt. protein and glucagon are discussed in the light of postulated mechanisms of glucagon biosynthesis.


[Tick borne zoonosis: selected clinical and diagnostic aspects].
Tick-borne zoonotic infections are among the most diffuse vector borne diseases: these large group of infections is caused by different microorganisms: Babesia spp., Borrelia spp., Rickettsia spp., Ehrlichia spp., Francisella tularensis, Coxiella burnetii) and tick-borne encephalitis virus. Babesiosis is caused by the protozoa (sporozoa) Babesia microti and it is quite rare in humans in Europe. The ixodids ticks are the competent vectors. A few symptomatic cases have been reported, mainly in splenectomized patients. The laboratory diagnosis is made by the microscopic identification of the parasites within the red blood cells in blood smears. The serologic diagnosis, based mainly upon IFA and WB techniques has only an epidemiological interest. Lyme borreliosis (Lyme disease) has been recognized as the most frequent vector borne disease in mild climate areas. The etiologic agent is a spirochete, belonging to the Borrelia burgdorferi sensu lato complex: B. burgdorferi sensu stricto, B. garinii and B. afzelii. Several additional species of this geno-complex have been identified but their pathogenic capability for humans still needs to be elucidated. Lyme borreliosis is clinically divided into three different clinical stages: the early disease, the disseminated infection and the persistent infection. Individual stages are caused by the diffusion of the spirochetes to different anatomic districts of the body. The main clinical symptoms are, for each stage: the erythema chronicum migrans in the early infection, the peripheral nerves and joint involvement in disseminated diseases and the acrodermatitis chronica atrophica (ACA) with central nervous system involvement in the late disseminated infection. The microbiological diagnosis is achieved by serologic techniques (IFA, EIA, WB) and by isolation of the spirochetes (in vitro culture and DNA amplification methods). Tick-borne relapsing fever (TBRF) is occasionally transmitted to humans by the soft ticks Ornithodorus and is caused by Borrelia spp. Different borreliae are responsible for TBRF in various geographic areas. The laboratory diagnosis is based upon the identification of spirochetes in peripheral blood by microscopic observation of Giemsa stained smears. Rickettsiosis diseases are caused worldwide by the obligate intracellular bacteria belonging to the genus Rickettsia. In the Mediterranean area the most frequently identified rickettsia is R. conorii, that causes the so called Mediterranean spotted fever. The serologic detection of a specific antibody response by IFA techniques is the most prominent tool for the diagnosis. In addition, the PCR method can be applied. Bacteria of the genus Ehrlichia are well known pathogens in veterinary medicine. Since the last decade their zoonotic capability has emerged and E. chafeensis, E. canis and the so called human granulocytic agent (HGE) have been identified in human diseases following a tick bite. The ehrlichiosis is characterized, in human, by a mild fever associated with lymphoadenopathy. The diagnosis is made on the identification of morulae (the intracytoplasmatic inclusion of the growing rickettsiae) in the white cells of peripheral blood. In addition the molecular diagnosis is also possible by PCR. Tick-borne encephalitis (TBE) is the only viral arthropod-borne encephalitis in Europe: it is caused by a flavivirus and it can also be transmitted by the ingestion of goat raw milk. The more relevant epidemiological figure is limited to the Alps, in particular to the Northern side (Austria). Isolated cases have been reported also in Italy. TBE is a benign self-limiting illness that usually recovers without any reliquate. The laboratory diagnosis is obtained by isolating the virus in cell cultures from the CSF or blood of acute phase patients. Serology is anyway the main laboratory tool to perform this diagnosis. Complement fixation and EIA IgM are the most used methods: the latter technique is particularly sensitive in early infection.


A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae.
A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.