Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Evaluation of wood characteristics of tropical post-mid rotation plantation Eucalyptus cloeziana and E. pellita: Part (c) Wood quality and structural properties.

This study provides information about wood quality, structural properties, processing characterists and product suitability of wood harvested from fast-grown hardwood plantations. Wood quality attributes tested included density, extractive content, unit shrinkage, heartwood proportion and sapwood width. Structural properties tested included small clear and full section strength and stiffness, hardness, joint group, visual grade assessment and natural vibration-based grade assessment. The variation between the inner, intermediate and outer heartwood zones and the variation between provenances was also tested. Overall, the wood qualtiy attributes measured for 19 year-old E. cloeziana and 15 year-old E. pellita plantation material fall between those expected from the wood of mature, native forest trees and those found in younger plantation material of the same species.

VEGF-C/VEGFR-3 and PDGF-B/PDGFR-b pathways in embryonic lymphangiogenesis

The circulatory system comprises the blood vascular system and the lymphatic vascular system. These two systems function in parallel. Blood vessels form a closed system that delivers oxygen and nutrients to the tissues and removes waste products from the tissues, while lymphatic vessels are blind-ended tubes that collect extravasated fluid and cells from the tissues and return them back to blood circulation. Development of blood and lymphatic vascular systems occurs in series. Blood vessels are formed via vasculogenesis and angiogenesis whereas lymphatic vessels develop via lymphangiogenesis, after the blood vascular system is already functional. Members of the vascular endothelial growth factor (VEGF) family are regulators of both angiogenesis and lymphangiogenesis, while members of the platelet-derived growth factor (PDGF) family are major mitogens for pericytes and smooth muscle cells and regulate formation of blood vessels. Vascular endothelial growth factor C (VEGF-C) is the major lymphatic growth factor and signaling through its receptor vascular endothelial growth factor receptor 3 (VEGFR-3) is sufficient for lymphangiogenesis in adults. We studied the role of VEGF-C in embryonic lymphangiogenesis and showed that VEGF-C is absolutely required for the formation of lymph sacs from embryonic veins. VEGFR-3 is also required for normal development of the blood vascular system during embryogenesis, as Vegfr3 knockout mice die at mid-gestation due to failure in remodeling of the blood vessels. We showed that sufficient VEGFR-3 signaling in the embryo proper is required for embryonic angiogenesis and in a dosage-sensitive manner for embryonic lymphangiogenesis. Importantly, mice deficient in both VEGFR-3 ligands, Vegfc and Vegfd, developed a normal blood vasculature, suggesting VEGF-C- and VEGF-D- independent functions for VEGFR-3 in the early embryo. Platelet-derived growth factor B (PDGF-B) signals via PDGFR-b and regulates formation of blood vessels by recruiting pericytes and smooth muscle cells around nascent endothelial tubes. We showed that PDGF-B fails to induce lymphangiogenesis when overexpressed in adult mouse skin using adenoviral vectors. However, mouse embryos lacking Pdgfb showed abnormal lymphatic vessels, suggesting that PDGF-B plays a role in lymphatic vessel maturation and separation from blood vessels during embryogenesis. Lymphatic vessels play a key role in immune surveillance, fat absorption and maintenance of fluid homeostasis in the body. However, lymphatic vessels are also involved in various diseases, such as lymphedema and tumor metastasis. These studies elucidate the basic mechanisms of embryonic lymphangiogenesis and add to the knowledge of lymphedema and tumor metastasis treatments by giving novel insights into how lymphatic vessel growth could be induced (in lymphedema) or inhibited (in tumor metastasis).

Precise Values of the "C Component Vicinal Coupling Constants for Calculating Side-Chain Conformations in Amino Acids

From consideration of 'H-lH vicinal coupling constants and '"G'H long-range coupling constants in a series of amino acid derivatives, the precise values of uC component vicinal coupling constants have been calculated for the three minimum energy staggered rotamers for the C(or)H-C(P)H, side-chains of amino acids.

Analyzing hysteresis behavior of capacitance-voltage characteristics of IZO/C₆₀/pentacene/Au diodes with a hole-transport electron-blocking polyterpenol layer by electric-field-induced optical second-harmonic generation measurement

By using electric-field-induced optical second-harmonic generation (EFISHG) measurement, we analyzed hysteresis behavior of capacitance-voltage (C-V) characteristics of IZO/polyterpenol (PT)/C₆₀/pentacene/Au diodes, where PT layer is actively working as a hole-transport electron-blocking layer. The EFISHG measurement verified the presence of interface accumulated charges in the diodes, and showed that a space charge electric field from accumulated excess electrons (holes) that remain at the PT/C₆₀ (C₆₀/pentacene) interface is responsible for the hysteresis loop observed in the C-V characteristics.

Evaluation of the impacts of spaying by either the dropped ovary technique or ovariectomy via flank laparotomy on the welfare of Bos indicus beef heifers and cows

The welfare outcomes for Bos indicus cattle (100 heifers and 50 cows) spayed by either the dropped ovary technique (DOT) or ovariectomy via flank laparotomy (FL) were compared with cattle subjected to physical restraint (PR), restraint by electroimmobilization in conjunction with PR (EIM), and PR and mock AI (MAI). Welfare assessment used measures of morbidity, mortality, BW change, and behavior and physiology indicative of pain and stress. One FL heifer died at d 5 from peritonitis. In the 8-h period postprocedures, plasma bound cortisol concentrations of FL, DOT, and EIM cows were not different and were greater (P < 0.05) than PR and MAI. Similarly, FL and DOT heifers had greater (P < 0.05) concentrations than PR and MAI, with EIM intermediate. Creatine kinase and aspartate aminotransferase concentrations were greater (P < 0.05) in FL and EIM heifers compared with the other treatments, with a similar pattern seen in the cows. Haptoglobin concentrations were significantly (P < 0.05) increased in the FL heifers compared with other treatments in the 8- to 24-h and 24- to 96-h periods postprocedures, and in cows were significantly (P < 0.05) increased in the FL and DOT compared with PR in the 24- to 96-h period. Behavioral responses complemented the physiological responses; standing head down was shown by more (P < 0.05) FL cows and heifers to 3 d postprocedures compared with other treatments, although there was no difference between FL and DOT heifers at the end of the day of procedures. At this same time, fewer (P < 0.05) FL and DOT heifers and cows were observed feeding compared with other treatments, although in cows there was no difference between FL, DOT, and EIM. There were no significant differences (P > 0.05) between treatments in BW changes. For both heifers and cows, FL and DOT spaying caused similar levels of acute pain, but FL had longer-lasting adverse impacts on welfare. Electroimmobilization during FL contributed to the pain and stress of the procedure. We conclude that: i) FL and DOT spaying should not be conducted without measures to manage the associated pain and stress; ii) DOT spaying is preferable to FL spaying; iii) spaying heifers is preferable to spaying cows; and iv) electroimmobilization causes pain and stress and should not be routinely used as a method of restraint.

Reciprocal and advanced generation hybrids between Corymbia citriodora and C-torelliana: forestry breeding and the risk of gene flow

Corymbia F1 hybrids have high potential for plantation forestry; however, little is known of their reproductive biology and potential for genetic pollution of native Corymbia populations. This study aims to quantify the influence of reproductive isolating barriers on the success of novel reciprocal and advanced generation Corymbia hybrids. Two maternal taxa, Corymbia citriodora subsp. citriodora and Corymbia torelliana, were pollinated using five paternal taxa, C. citriodora subsp. citriodora, C. torelliana, one C. torelliana x C. citriodora subsp. citriodora hybrid and two C. torelliana x C. citriodora subsp. variegata hybrids. Pollen tube, embryo and seed development were assessed. Reciprocal hybridisation between C. citriodora subsp. citriodora and C. torelliana was successful. Advanced generation hybrids were also created when C. citriodora subsp. citriodora or C. torelliana females were backcrossed with F1 hybrid taxa. Prezygotic reproductive isolation was identified via reduced pollen tube numbers in the style and reduced numbers of ovules penetrated by pollen tubes. Reproductive isolation was weakest within the C. citriodora subsp. citriodora maternal taxon, with two hybrid backcrosses producing equivalent capsule and seed yields to the intraspecific cross. High hybridising potential was identified between all Corymbia species and F1 taxa studied. This provides opportunities for advanced generation hybrid breeding, allowing desirable traits to be amplified. It also indicates risks of gene flow between plantation and native Corymbia populations.

Novel intramolecular reductive C-C coupling during borohydride reduction of a bi-enone

Borohydride reduction of the bi-enone (1) gave the structurally and mechanistically interesting compounds (4) and (5a-c) resulting from intramolecular carbon-carbon coupling.

Structure of l-Diphenylmethyl-3-hydroxyazetidinium Chloride,* C 16 H lsN O+.CI -

M r=275.8, monoclinic, P21/a, a= 12.356 (5), b=9.054 (4), c= 14.043 (4) A, t= 100.34 (3) ° , V=1545.5A 3, Z=4, D,,,= 1.14, D x = 1.185 Mg m -3, p(Mo Ka, /l = 0.7107 ]k) = 2.77 mm -1, F(000) = 584.0, T= 293 K, R = 0.053 for 1088 reflections. The four-membered ring is buckled 13.0 ° (0= 167.0°). The azetidinium moiety is linked to the C1- ion through a hydrogen bond [O-H...C1 = 3.166 (5) A].

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana × C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.

Preservation of encapsulated shoot tips and nodes of the tropical hardwoods Corymbia torelliana × C. citriodora and Khaya senegalensis

Alginate encapsulation is a simple and cost-effective technique to preserve plant germplasm but there are only a few reports available on preservation of encapsulated explants of two highly valuable groups of tropical trees, the eucalypts (Myrtaceae) and mahoganies (Meliaceae). This study investigated alginate encapsulation for preservation of the eucalypt hybrid, Corymbia torelliana × C. citriodora, and the African mahogany, Khaya senegalensis. We assessed shoot regrowth of encapsulated shoot tips and nodes after storage for 0, 3, 6 and 12 months on media varying in sucrose and nutrient content, under storage conditions of 14°C and zero-irradiance. Encapsulated explants of both trees were preserved most effectively on high-nutrient (half-strength Murashige and Skoog) medium containing 1% sucrose, which provided very high frequencies of shoot regrowth (92–100% for Corymbia and 71–98% for Khaya) and excellent shoot development after 12 months’ storage. This technique provides an extremely efficient means for storage and exchange of eucalypts and mahoganies, ideally suited for incorporation into plant breeding and germplasm conservation programs.

Physiological and behavioral responses of sheep to gaseous ammonia

Ammonia can accumulate in highly stocked sheep accommodation, for example during live export shipments, and could affect sheep health and welfare. Thus, the objective of this experiment was to test the effects of 4 NH3 concentrations, 4 (control), 12, 21, and 34 mg/m(3), on the physiology and behavior of wether sheep. Sheep were held for 12 d under a micro-climate and stocking density similar to shipboard conditions recorded on voyages from Australia to the Middle East during the northern hemispheric summer. Ammonia increased macrophage activity in transtracheal aspirations, indicating active pulmonary infl ammation; however, it had no effect (P > 0.05) on hematological variables. Feed intake decreased (P = 0.002) in proportion to ammonia concentration, and BW gain decreased (P < 0.001) at the 2 greatest concentrations. Exposure to ammonia increased (P = 0.03) the frequency of sneezing, and at the greatest ammonia concentration, sheep were less active, with less locomotion, pawing, and panting. Twenty-eight days after exposure to NH3, the pulmonary macrophage activity and BW of the sheep returned to that of sheep exposed to only 4 mg/m(3). It was concluded that NH3 induced a temporary inflammatory response of the respiratory system and reduced BW gain, which together indicated a transitory adverse effect on the welfare of sheep.