Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis

We describe the synthesis and characterization of a 5′ conjugate between a 2′-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the αvβ3 integrin. We used αvβ3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD–623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while ‘free’ 623 did not. However, the kinetics of luciferase expression was distinct; the RGD–623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD–623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD–623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide–oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the αvβ3 integrin

Physicochemical properties and structural characteristics of whole grain Oryza sativa L. with different treatments

[EN] Physicochemical properties and structural characteristics of whole rice flours with different treatments (soaking, germination and extrusion cooking) were studied. Water solubility, water absorption, crystallinity, adsorption isotherms (BET and GAB models), and glass transition temperature of the samples were determined. Water solubility and water absorption were enhanced by extrusion cooking process (3.17 4.98 vs. 24.1 53.76 g/100 g and 2.77 3.05 vs. 4.46 7.04 ml/g, respectively), but crystallinity was decreased (30 33 vs. 4 16%). Adsorption isotherms showed that extruded samples exhibited higher equilibrium moisture content as compared with their corresponding non-extruded samples (5.0 19.2 vs. 4.0 16.1 g water/g solids). There were no changes in glass transition temperature values in the studied moisture range (3.8 16 g/100 g). These results allow the correct use of whole rice flours with different treatments in foods and also contributed to the knowledge of stabilization of the productsThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was partially financed by ANPCYT (PICT 1105) and ERASMUS MUNDUS ACTION 2 ARCOIRIS Fellowship.Albarracin, M.; Talens Oliag, P.; Martínez Navarrete, N.; González, RJ.; Drago, SR. (2016). Physicochemical properties and structural characteristics of whole grain Oryza sativa L. with different treatments. Food Science and Technology International. 22(4):1-10. doi:10.1177/1082013215600078S110224Albarracín, M., José González, R., & Drago, S. R. (2015). Soaking and extrusion effects on physicochemical parameters, phytic acid, nutrient content and mineral bio-accessibility of whole rice grain. International Journal of Food Sciences and Nutrition, 66(2), 210-215. doi:10.3109/09637486.2014.986070Björck, I., & Asp, N.-G. (1983). The effects of extrusion cooking on nutritional value — A literature review. Journal of Food Engineering, 2(4), 281-308. doi:10.1016/0260-8774(83)90016-xBrunauer, S., Deming, L. S., Deming, W. E., & Teller, E. (1940). On a Theory of the van der Waals Adsorption of Gases. Journal of the American Chemical Society, 62(7), 1723-1732. doi:10.1021/ja01864a025Donkor, O. N., Stojanovska, L., Ginn, P., Ashton, J., & Vasiljevic, T. (2012). Germinated grains – Sources of bioactive compounds. Food Chemistry, 135(3), 950-959. doi:10.1016/j.foodchem.2012.05.058Gonzalez, R. J., De Greef, D. M., Torres, R. L., Borras, F. S., & Robutti, J. (2004). Effects of endosperm hardness and extrusion temperature on properties of products obtained with grits from two commercial maize cultivars. LWT - Food Science and Technology, 37(2), 193-198. doi:10.1016/j.lwt.2003.07.004Gonzalez, R., Drago, S., Torres, R., & De Greef, D. (2013). Extrusion Cooking of Cereal-Based Products. Contemporary Food Engineering. doi:10.1201/b15246-13González, R. J., Pastor Cavada, E., Vioque Peña, J., Torres, R. L., De Greef, D. M., & Drago, S. R. (2013). Extrusion Conditions and Amylose Content Affect Physicochemical Properties of Extrudates Obtained from Brown Rice Grains. International Journal of Food Science, 2013, 1-8. doi:10.1155/2013/584148Herawat, H., Kusnandar, F., Adawiyah, D. R., Budijanto, S., & Rahman, M. S. (2014). Thermal characteristics and state diagram of extruded instant artificial rice. Thermochimica Acta, 593, 50-57. doi:10.1016/j.tca.2014.08.017Jones, J. M., & Engleson, J. (2010). Whole Grains: Benefits and Challenges. Annual Review of Food Science and Technology, 1(1), 19-40. doi:10.1146/annurev.food.112408.132746Kim, H. Y., Hwang, I. G., Kim, T. M., Woo, K. S., Park, D. S., Kim, J. H., … Jeong, H. S. (2012). Chemical and functional components in different parts of rough rice (Oryza sativa L.) before and after germination. Food Chemistry, 134(1), 288-293. doi:10.1016/j.foodchem.2012.02.138Lowry, R. R., & Tinsley, I. J. (1976). Rapid colorimetric determination of free fatty acids. Journal of the American Oil Chemists’ Society, 53(7), 470-472. doi:10.1007/bf02636814Matveev, Y. (2000). The plasticizing effect of water on proteins, polysaccharides and their mixtures. Glassy state of biopolymers, food and seeds. Food Hydrocolloids, 14(5), 425-437. doi:10.1016/s0268-005x(00)00020-5Perdon, A., Siebenmorgen, T. J., & Mauromoustakos, A. (2000). Glassy State Transition and Rice Drying: Development of a Brown Rice State Diagram. Cereal Chemistry Journal, 77(6), 708-713. doi:10.1094/cchem.2000.77.6.708ROOS, Y., & KAREL, M. (1991). Plasticizing Effect of Water on Thermal Behavior and Crystallization of Amorphous Food Models. Journal of Food Science, 56(1), 38-43. doi:10.1111/j.1365-2621.1991.tb07970.xRuiz-Ruiz, J., Martínez-Ayala, A., Drago, S., González, R., Betancur-Ancona, D., & Chel-Guerrero, L. (2008). Extrusion of a hard-to-cook bean (Phaseolus vulgaris L.) and quality protein maize (Zea mays L.) flour blend. LWT - Food Science and Technology, 41(10), 1799-1807. doi:10.1016/j.lwt.2008.01.005SIU, G. M., & DRAPER, H. H. (1978). A SURVEY OF THE MALONALDEHYDE CONTENT OF RETAIL MEATS AND FISH. Journal of Food Science, 43(4), 1147-1149. doi:10.1111/j.1365-2621.1978.tb15256.xSun, Z., Yang, W., Siebenmorgen, T., Stelwagen, A., & Cnossen, A. (2002). Thermomechanical Transitions of Rice Kernels. Cereal Chemistry Journal, 79(3), 349-353. doi:10.1094/cchem.2002.79.3.349Timmermann, E. O. (2003). Multilayer sorption parameters: BET or GAB values? Colloids and Surfaces A: Physicochemical and Engineering Aspects, 220(1-3), 235-260. doi:10.1016/s0927-7757(03)00059-1Tovar, J., Bjoerck, I. M., & Asp, N. G. (1990). Starch content and .alpha.-amylolysis rate in precooked legume flours. Journal of Agricultural and Food Chemistry, 38(9), 1818-1823. doi:10.1021/jf00099a00

Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars

<p>Abstract</p> <p>Background</p> <p>Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds using both massively parallel signature sequencing (MPSS) and sequencing-by-synthesis (SBS). Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield), LaGrue (low milling yield), Ilpumbyeo (high eating quality), YR15965 (low eating quality), and Nipponbare (control).</p> <p>Results</p> <p>The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90), and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved <it>cis </it>regulatory elements were identified. Numerous specifically expressed transcription factor (TF) genes were identified in Cypress (282), LaGrue (312), Ilpumbyeo (363), YR15965 (260), and Nipponbare (357). Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation) that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase) and granule bound starch synthase I (GBSS I) in Cypress than that in LaGrue during early seed development.</p> <p>Conclusion</p> <p>This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved in the biosynthesis of starch, aspartate family amino acids, and storage proteins. Some of the differentially expressed genes could be useful for the development of molecular markers if they are located in a known QTL region for milling yield or eating quality in the rice genome. Therefore, our comprehensive and deep survey of the developing seed transcriptome in five rice cultivars has provided a rich genomic resource for further elucidating the molecular basis of grain quality in rice.</p

How Much Remains Undetected? Probability of Molecular Detection of Human Plasmodia in the Field

BACKGROUND: In malaria endemic areas, most people are simultaneously infected with different parasite clones. Detection of individual clones is hampered when their densities fluctuate around the detection limit and, in case of P. falciparum, by sequestration during part of their life cycle. This has important implications for measures of levels of infection or for the outcome of clinical trials. This study aimed at measuring the detectability of individual P. falciparum and P. vivax parasite clones in consecutive samples of the same patient and at investigating the impact of sampling strategies on basic epidemiological measures such as multiplicity of infection (MOI). METHODS: Samples were obtained in a repeated cross-sectional field survey in 1 to 4.5 years old children from Papua New Guinea, who were followed up in 2-monthly intervals over 16 months. At each follow-up visit, two consecutive blood samples were collected from each child at intervals of 24 hours. Samples were genotyped for the polymorphic markers msp2 for P. falciparum and msp1F3 and MS16 for P. vivax. Observed prevalence and mean MOI estimated from single samples per host were compared to combined data from sampling twice within 24 h. FINDINGS AND CONCLUSION: Estimated detectability was high in our data set (0.79 [95% CI 0.76-0.82] for P. falciparum and, depending on the marker, 0.61 [0.58-0.63] or 0.73 [0.71-0.75] for P. vivax). When genotyping data from sequential samples, collected 24 hours apart, were combined, the increase in measured prevalence was moderate, 6 to 9% of all infections were missed on a single day. The effect on observed MOI was more pronounced, 18 to 31% of all individual clones were not detected in a single bleed. Repeated sampling revealed little difference between detectability of P. falciparum and P. vivax

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase&nbsp;1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation&nbsp;disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age&nbsp; 6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score&nbsp; 652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc&nbsp;= 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N&nbsp;= 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in&nbsp;Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in&nbsp;Asia&nbsp;and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701