Added Value of Next Generation over Sanger Sequencing in Kenyan Youth with Extensive HIV-1 Drug Resistance.

HIV-1 drug resistance testing in children and adolescents in low-resource settings is both important and challenging. New (more sensitive) drug resistance testing technologies may improve clinical care, but evaluation of their added value is limited. We assessed the potential added value of using next-generation sequencing (NGS) over Sanger sequencing for detecting nucleoside reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations (DRMs). Participants included 132 treatment-experienced Kenyan children and adolescents with diverse HIV-1 subtypes and with already high levels of drug resistance detected by Sanger sequencing. We examined overall and DRM-specific resistance and its predicted impact on antiretroviral therapy and evaluated the discrepancy between Sanger sequencing and six NGS thresholds (1%, 2%, 5%, 10%, 15%, and 20%). Depending on the NGS threshold, agreement between the two technologies was 62% to 88% for any DRM, 83% to 92% for NRTI DRMs, and 73% to 94% for NNRTI DRMs, with more DRMs detected at low NGS thresholds. NGS identified 96% to 100% of DRMs detected by Sanger sequencing, while Sanger identified 83% to 99% of DRMs detected by NGS. Higher discrepancy between technologies was associated with higher DRM prevalence. Even in this resistance-saturated cohort, 12% of participants had higher, potentially clinically relevant predicted resistance detected only by NGS. These findings, in a young, vulnerable Kenyan population with diverse HIV-1 subtypes and already high resistance levels, suggest potential benefits of more sensitive NGS over existing technology. Good agreement between technologies at high NGS thresholds supports their interchangeable use; however, the significance of DRMs identified at lower thresholds to patient care should be explored further. IMPORTANCE HIV-1 drug resistance in children and adolescents remains a significant problem in countries facing the highest burden of the HIV epidemic. Surveillance of HIV-1 drug resistance in children and adolescents is an important public health strategy, particularly in resource-limited settings, and yet, it is limited due mostly to cost and infrastructure constraints. Whether newer and more sensitive next-generation sequencing (NGS) adds substantial value beyond traditional Sanger sequencing in detecting HIV-1 drug resistance in real life settings remains an open and debatable question. In this paper, we attempt to address this issue by performing a comprehensive comparison of drug resistance identified by Sanger sequencing and six NGS thresholds. We conducted this study in a well-characterized, vulnerable cohort of children and adolescents living with diverse HIV-1 subtypes in Kenya and, importantly, failing antiretroviral therapy (ART) with already extensive drug resistance. Our findings suggest a potential added value of NGS over Sanger even in this unique cohort.

Evolution of the grass leaf by primordium extension and petiole-lamina remodeling.

The sheathing leaf found in grasses and other monocots is an evolutionary innovation, yet its origin has been a subject of long-standing debate. Here, we revisit the problem in the light of developmental genetics and computational modeling. We show that the sheathing leaf likely arose through WOX-gene-dependent extension of a primordial zone straddling concentric domains around the shoot apex. Patterned growth within this zone, oriented by two polarity fields, accounts for wild-type, mutant and mosaic grass leaf development, whereas zone contraction and growth remodeling accounts for eudicot leaf development. In contrast to the prevailing view, our results suggest that the sheath derives from petiole, whereas the blade derives from the lamina of the eudicot leaf, consistent with homologies proposed in the 19th century.

Substituted 1-methyl-4-phenylpyrrolidin-2-ones - Fragment-based design of N-methylpyrrolidone-derived bromodomain inhibitors.

N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.

Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition.

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.

Pulse Flour Characteristics from a Wheat Flour Miller's Perspective: A Comprehensive Review.

Pulses (grain legumes) are increasingly of interest to the food industry as product formulators and consumers seek to exploit their fiber-rich and protein-rich reputation in the development of nutritionally attractive new products, particularly in the bakery, gluten-free, snack, pasta, and noodle categories. The processing of pulses into consistent high-quality ingredients starts with a well-defined and controlled milling process. However, in contrast to the extensive body of knowledge on wheat flour milling, the peer-reviewed literature on pulse flour milling is not as well defined, except for the dehulling process. This review synthesizes information on milling of leguminous commodities such as chickpea (kabuli and desi), lentil (green and red), pea, and bean (adzuki, black, cowpea, kidney, navy, pinto, and mung) from the perspective of a wheat miller to explore the extent to which pulse milling studies have addressed the objectives of wheat flour milling. These objectives are to reduce particle size (so as to facilitate ingredient miscibility), to separate components (so as to improve value and/or functionality), and to effect mechanochemical transformations (for example, to cause starch damage). Current international standards on pulse quality are examined from the perspective of their relationship to the millability of pulses (that is, grain legume properties at mill receival). The effect of pulse flour on the quality of the products they are incorporated in is examined solely from the perspective of flour quality not quantity. Finally, we identify research gaps where critical questions should be answered if pulse milling science and technology are to be established on par with their wheat flour milling counterparts.

Bubbles in noodle dough: Characterization by X-ray microtomography.

Bubbles, found in a huge variety of food products, are known to afford desirable quality attributes, especially those related to texture, mouthfeel and taste. However, the presence of bubbles and their effects on wheat flour noodles is an aspect that has been, until now, largely overlooked, despite the positive and negative connotations of bubbly inclusions on Asian noodle quality. X-rays from a synchrotron source (Biomedical Imaging and Therapy facility at the Canadian Light Source) were used to rapidly and non-destructively acquire tomographic images of noodle dough. Appropriate image analysis protocols were used to determine the bubble size distribution, the orientation of bubbles, and their position within the dough sheet. The effect of processing (one or multiple lamination steps) on bubble properties in the dough that was subsequently sheeted (gradual elongation and reduction in thickness) was investigated. Bubble size distributions, well captured by lognormal distribution function, showed that the lamination process induced bubble entrapment and reduction in bubble size. Bubbles were found to be flat, elongated and oriented in the sheeting direction, this effect being less for doughs laminated ten times (90° rotations between lamination steps). Interestingly, a gradient in concentration of bubbles within the dough sheet was found from the noodle core to the sheet edges. Aging effects were also apparent. This first non-destructive study of bubbles in wheat-flour noodle dough provides a more complete knowledge of the dough sheet's internal structure, and how it originates via processing, and this has repercussions on the overall quality of Asian noodles.

Development of emulsifying property in Persian gum using octenyl succinic anhydride (OSA).

In the present study, the influence of octenyle succinic anhydride (OSA),gum concentration, pH, temperature and reaction time on esterification of Persian gum (PG), and its soluble (SFPG) and insoluble (IFPG) fractions, were investigated by response surface methodology (RSM) in order to optimize the reaction conditions based on the degree of substitution (DS). The individual effect of all independent variables as well as the interactive effects of temperature-OSA concentration, and OSA-PG concentrations on DS was significant. However, the latter interactive effect (OSA-SFPG) was not significant in case of SFPG. The IFPG did not have any esterification reaction with OSA. The highest DS for PG and SFPG were 0.0285 and 0.0303 at the optimal conditions, respectively. The FTIR spectrums also confirmed the carbonyl group attachment in OSA-PG and OSA-SFPG. The enhancement of emulsifying capability was also confirmed by ECI and EAI values, microscopic images as well as rheological measurements.

Influence of internal interfacial area on nanosecond relaxation of wheat gluten proteins as probed by broadband ultrasonic spectroscopy.

Understanding interactions between interfaces and biopolymers in complex industrially processed materials of plant origin will allow for their better utilization. Wheat flour doughs are one such material whose industrial use strongly depends on such interactions due to their effect on the mechanical properties of the dough. To date, mechanical characterizations of dough have been limited to a narrow range of frequencies. Here, ultrasonic spectroscopy measurements over a very broad frequency range are used to show that a fast volumetric relaxation occurs in dough; the nanosecond timescale of the relaxation is associated with ultrasonic stress-induced changes in the secondary structure of gluten proteins. Interestingly, there is a four-fold difference in the speed of this relaxation phenomenon in doughs mixed in air (where substantial internal interfacial area exists) compared to those mixed under vacuum (where bubbles are absent). Given the large internal interfacial area in dough, the amphiphilic proteins residing at gas bubble interfaces significantly alter the high-frequency mechanical response of this important material.

Probing thermal transitions and structural properties of gluten proteins using ultrasound.

PURPOSE: To probe the thermal and structural properties of gluten proteins using ultrasound. METHODS: A new ultrasonic approach for characterizing the quality of wheat gluten proteins is described. Low frequency (50 kHz) longitudinal ultrasonic velocity, v L, measurements were performed on gluten samples extracted from three wheat flours differing in protein content and in wheat endosperm hardness. RESULTS: At room temperature, v L for gluten extracted from soft flowers (Fielder) was found to be (870 ± 92) m/s, while for gluten extracted from extra strong flours (Glenlea) it was found to be (1,940 ± 90) m/s. In the second set of experiments, which aimed at probing thermal properties of gluten proteins, the variation in the numerical value of v L propagating in the wet gluten was found to be substantial (about 1,000 m/s) when the temperature of the gluten was raised from 20 to 90 °C, and also when gluten from different flour types was investigated. A continuous structural phase transition was observed, which was different for glutens extracted from different flours. Upon cooling, the velocity also varied depending on wheat type. CONCLUSIONS: These experiments demonstrate that ultrasonic velocity measurements can be used as a selection tool and study changes in properties of wheat proteins, particularly the thermal transitions that are critical to the quality of end products such as noodles, pasta, and bread. It was also shown that v L is sensitive to gluten class (strength or protein content), showing the potential of such measurements as an early-generation selection tool in wheat breeding programs.

Reducing catheter-associated urinary tract infections in home care: a performance improvement project.

The Performance Improvement Department of one home healthcare agency (HHA) identified an increase in the rate of catheter-associated urinary tract infections (CAUTI) during 2009. An investigation was undertaken to identify factors that contributed to this increase and an action plan was implemented to reduce the rate of infections. Modifications were made to the surveillance process to align the infection rate calculation with Centers for Disease Control and Prevention (CDC) guidelines and staff education was undertaken to ensure utilization of evidence-based practice. An overall reduction in the CAUTI rate was achieved through this multifactorial approach.

Transmission of ultrasound through a single layer of bubbles.

We investigate, both experimentally and theoretically, the effect of coupling between resonant scatterers on the transmission coefficient of a model system of isotropic scatterers. The model system consists of a monodisperse layer of bubbles, which exhibit a strong monopole scattering resonance at low ultrasonic frequencies. The layer was a true 2D structure obtained by injecting very monodisperse bubbles (with radius a approximately 100 microm) into a yield-stress polymer gel. Even for a layer with a low concentration of bubbles (areal fraction, n pi a(2), of 10-20%, where n is the number of bubbles per unit area), the ultrasonic transmission was found to be significantly reduced by the presence of bubbles (-20 to -50 dB) and showed a sharp minimum at a particular frequency. Interestingly, this frequency did not correspond to the resonance frequency of the individual, isolated bubbles, but depended markedly on the concentration. This frequency shift is an indication of strong coupling between the bubbles. We propose a simple model, based on a self-consistent relation, which takes into account the coupling between the bubbles and gives good agreement with the measured transmission coefficient.

Ultrasonic investigation of the effect of vegetable shortening and mixing time on the mechanical properties of bread dough.

Mixing is a critical stage in breadmaking since it controls gluten development and nucleation of gas bubbles in the dough. Bubbles affect the rheology of the dough and largely govern the quality of the final product. This study used ultrasound (at a frequency where it is sensitive to the presence of bubbles) to nondestructively examine dough properties as a function of mixing time in doughs prepared from strong red spring wheat flour with various amounts of shortening (0%, 2%, 4%, 8% flour weight basis). The doughs were mixed for various times at atmospheric pressure or under vacuum (to minimize bubble nucleation). Ultrasonic velocity and attenuation (nominally at 50 kHz) were measured in the dough, and dough density was measured independently from specific gravity determinations. Ultrasonic velocity decreased substantially as mixing time increased (and more bubbles were entrained) for all doughs mixed in air; for example, in doughs made without shortening, velocity decreased from 165 to 105 ms(-1), although superimposed on this overall decrease was a peak in velocity at optimum mixing time. Changes in attenuation coefficient due to the addition of shortening were evident in both air-mixed and vacuum-mixed doughs, suggesting that ultrasound was sensitive to changes in the properties of the dough matrix during dough development and to plasticization of the gluten polymers by the shortening. Due to its ability to probe the effect of mixing times and ingredients on dough properties, ultrasound has the potential to be deployed as an online quality control tool in the baking industry.

Pheochromocytoma in Pregnancy: When is Operative Intervention Indicated?

Pheochromocytoma is rare in pregnancy, with an estimated incidence of 0.007%. Diagnosis is difficult owing to the variety of presentations and nonspecific symptoms. Nevertheless, unsuspected disease accounts for a significant proportion of morbidity and mortality. Currently, there appears to be no consensus on management with regard to the need for and timing of medical vs. surgical management. In this case report, we describe two patients who underwent different modes of treatment based on careful consideration of disease-related and nondisease-related factors. We emphasise that good outcomes can be achieved through individualized management within the context of a multidisciplinary team, involving close collaboration among physicians, surgeons, obstetricians, and anesthetists. We also illustrate the importance of genetic testing in all patients with pheochromocytoma in pregnancy, especially with the emergence of new predisposing genes (succinate dehydrogenase B and D) and the recognition that germline mutations in these and more established genes (VHL and RET) account for over a quarter of all apparently sporadic cases.

Extrusion of pea starch containing lysozyme and determination of antimicrobial activity.

Pea starch, which has inherently good gel strength, was used as the source material for manufacturing a biodegradable and bioactive packaging material. Extrudates containing 99% pea starch and 1% lysozyme were produced under various extrusion conditions (high and low shear screw configurations, 30% to 40% moisture contents, 70 to 150 degrees C die temperatures). The physical and mechanical properties of the extrudates were determined through various expansion indices, piece and cell wall solid density, compression, and 3-point bending tests. The expansion of extrudates increased with an increase in die temperature, whereas increasing moisture content had the opposite effect. Extrudate densities decreased as extrusion temperature increased, whereas lower moisture content in the extrudate dough decreased extrudate densities. The elastic modulus and fracture strengths were highly correlated in a power-law fashion to relative density, showing that the mechanical properties of extrudates were dependent on solid density and foam structure. Up to 48% of the initial lysozyme activity was recovered from the extruded pea starch matrix. The lysozyme released from extrudates showed an inhibition zone against Brochotrix thermosphacta B2. Extruded pea starch matrix containing lysozyme has potential application as an edible and biodegradable packaging material with antimicrobial activity.

Factors affecting river health and its assessment over broad geographic ranges: the Western Australian experience.

AusRivAS is an Australia-wide program that measures river condition using predictive models to compare the macroinvertebrate families occurring at a river site with those expected if the site were in natural condition. Results of assessment of 685 sites across all major rivers in Western Australia are presented. Most rivers were in relatively natural condition in the northern half of the state where the human population is low and pastoralism is the major land use. In the south, where the human population is higher and agriculture is more intensive, rivers were mostly more disturbed. AusRivAS assessment produced some erroneous results in rivers of the south-west cropping zone because of the lack of appropriate reference site groups and biased distribution of sampling sites. Collecting low numbers of animals from many forested streams, because of low stream productivity and samples that were difficult to sort, also affected assessments. Overall, however, AusRivAs assessment identified catchment processes that were inimical to river health. These processes included salinisation, high nutrient and organic loads, erosion and loss of riparian vegetation. River regulation, channel modification and fire were also associated with river degradation. As is the case with other assessment methods, one-off sampling at individual sites using AusRivAS may be misleading. Seasonal drought, in particular, may make it difficult to relate conditions at the time of sampling to longer-term river health. AusRivAS has shown river condition in Western Australia is not markedly different from other parts of Australia which, as a whole, lacks the substantial segments of severely degraded river systems reported in England.

Characterizing a model food gel containing bubbles and solid inclusions using ultrasound.

Ultrasonic spectroscopy is used to characterize a model aerated food system consisting of agar gel in which both bubbles and polystyrene beads are embedded. By exploiting the distinct frequency dependence of each inclusion's acoustic resonances, it is demonstrated that the sizes of the bubbles and beads can be measured by ultrasound even when the size distributions are so similar that these inclusions are difficult to distinguish in optical images. While these results demonstrate the potential for applying ultrasonic spectroscopy to evaluate any soft heterogeneous material in which both bubbles and solid inclusions are present, the technique is especially relevant for functional foods, in which solid functional ingredients must be incorporated without degrading the aerated structure of the food and causing unacceptable quality impairment.

Increasing thyroxine requirements in primary hypothyroidism: don't forget the urinalysis!

Rising thyroid stimulating hormone (TSH) levels in patients being treated for primary hypothyroidism usually indicate poor compliance with thyroxine therapy. In rare instances, drugs or diseases affecting absorption of thyroxine or drugs that accelerate thyroxine metabolism can manifest in a similar fashion. Nephrotic syndrome is a rare cause of such a presentation though its presence can rapidly be suspected by dipstick urine testing. In this report we describe a patient with long-standing primary thyroid failure whose thyroxine dose requirements increased upon development of massive proteinuria. Biochemical testing and renal biopsy subsequently demonstrated nephrotic syndrome and amyloid deposition in association with myeloma. Dipstick urine testing should be considered in all hypothyroid patients with rising TSH levels, where good compliance with thyroxine therapy is likely.

Baseline characteristics and response to GH replacement of hypopituitary patients previously irradiated for pituitary adenoma or craniopharyngioma: data from the Pfizer International Metabolic Database.

OBJECTIVE: To test the hypothesis whether the effects of GH replacement therapy in adults could be affected by prior pituitary irradiation, the baseline characteristics and response to GH were evaluated in adults with severe GH deficiency (GHD), who had received or not irradiation for the treatment of pituitary adenoma or craniopharyngioma. DESIGN: Data from 447 patients, who had received radiotherapy (427 in addition to surgery), and 630 patients, who were operated on but not irradiated for their tumour, were retrieved from Pfizer International Metabolic Database (KIMS) and compared at baseline and 1 and 2 years following the onset of GH replacement. RESULTS: Irradiated and non-irradiated patients exhibited the expected phenotype of GHD at baseline. However, irradiated patients had a greater impairment in the quality of life (QoL), a higher fat mass, lower high-density lipoprotein cholesterol levels and a lower bone mineral content (BMC) than non-irradiated patients. Treatment with GH induced similar changes in both groups. After 1 year of GH replacement, there was an increase in serum IGF-I and fat-free mass, a reduction in fat mass and an improvement in QoL, all changes being equivalent in irradiated and non-irradiated patients. The lipid profile also improved with the irradiated patients showing a better response. These beneficial effects were maintained and the BMC also increased in both groups by the second year of treatment. CONCLUSIONS: This analysis shows that prior irradiation for pituitary adenoma or craniopharyngioma does not compromise the beneficial effects of GH replacement therapy.

Impaired vasoreactivity in bodybuilders using androgenic anabolic steroids.

BACKGROUND: Anabolic androgenic steroids are used by some bodybuilders to enhance performance. While the cardiovascular implications of supraphysiological androgen levels requires further clarification, use is associated with sudden death, left ventricular hypertrophy, thrombo-embolism and cerebro-vascular events. MATERIALS AND METHODS: To further understand the effect of androgenic anabolic steroid abuse on vascular function, this study assessed vascular stiffness (pulse-wave analysis) and cardiovascular risk factors in 28 male, bodybuilding subjects, of whom ten were actively receiving anabolic agents (group A; 26.4 +/- 7.2 years) and eight had undergone a 3-month "wash-out" period (group B; 32.1 +/- 7.1 years). The remaining ten bodybuilding subjects (group C; 24.4 +/- 4.4 years) denied any past use of anabolic steroids or other performance enhancing drugs. Comparisons were made with ten sedentary male controls (group D, 29.3 +/- 4.7 years). RESULTS: Endothelial independent dilatation in response to glycerol trinitrate was significantly impaired in the group currently using anabolic steroids (group A) compared with the other three groups [A (5.63 +/- 3.24%) versus; B (11.10 +/- 4.91%), C (17.88 +/- 9.2%) and D (14.46 +/- 3.9%), P < 0.0005, respectively], whereas no significant differences in endothelial-dependent dilatation were detected between the groups [A (5.0 +/- 3.0%), B (7.4 +/- 3.4%), C (9.6 +/- 4.5%) and D (8.2 +/- 3.3%), P < 0.059, respectively]. CONCLUSIONS: Previous studies described a decline in vascular reactivity occurring in bodybuilding subjects which is independent of anabolic steroid use and may result from smooth muscle hypertrophy with increased vascular stiffness. This study revealed impaired vascular reactivity associated with anabolic agents and that improvement in vascular function may occur following their discontinuation.

Medical injuries among hospitalized children.

BACKGROUND: Inpatient medical injuries among children are common and result in a longer stay in hospital and increased hospital charges. However, previous studies have used screening criteria that focus on inpatient occurrences only rather than on injuries that also occur in ambulatory or community settings leading to hospital admission. OBJECTIVE: To describe the incidence and outcomes of medical injuries among children hospitalized in Wisconsin using the Wisconsin Medical Injury Prevention Program (WMIPP) screening criteria. METHODS: Cross sectional analysis of discharge records of 318,785 children from 134 hospitals in Wisconsin between 2000 and 2002. RESULTS: The WMIPP criteria identified 3.4% of discharges as having one or more medical injuries: 1.5% due to medications, 1.3% to procedures, and 0.9% to devices, implants and grafts. After adjusting for the All Patient Refined-Diagnosis Related Groups disease category, illness severity, mortality risk, and clustering within hospitals, the mean length of stay (LOS) was a half day (12%) longer for patients with medical injuries than for those without injuries. The similarly adjusted mean total hospital charges were 1614 dollars (26%) higher for the group with medical injuries. Excess LOS and charges were greatest for injuries due to genitourinary devices/implants, vascular devices, and infections/inflammation after procedures. CONCLUSIONS: This study reinforces previous national findings up to 2000 using Wisconsin data to the end of 2002. The results suggest that hospitals and pediatricians should focus clinical improvement on medications, procedures, and devices frequently associated with medical injuries and use medical injury surveillance to track medical injury rates in children.