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There has been increasing interest over recent years in the application of three-dimensional (3D) printing technologies in the biomedical field. One such method is Direct Ink Writing (DIW); this approach has the potential advantage of allowing room-temperature deposition of materials, presented as an ink, to build complex architectures. DIW offers the ability to process biomaterials containing temperature-sensitive components. Due to the fabrication principles of DIW, there are specific rheological requirements that the ink must exhibit for the 3D construction. For this reason, hydrogel-based liquid feed stocks have been the focal point of ink development. As a consequence, studies based on inks comprising hydrophobic biomaterials, which are insoluble in water and hence unsuited to the hydrogel approach, have been limited. In this study, we investigate novel inks that utilize polycaprolactone (PCL), a hydrophobic polymer, as the primary constituent by dissolving the polymer in solvent systems based on dichloromethane (DCM) and acetone (ACE). Moreover, polyethylene oxide (PEO) was incorporated into the PCL systems in order to extend the range of hydrophilicity of the systems. The rheological properties of the inks were investigated as a function of polymer composition and solvent system. Woodpile constructs of PCL and PCL/PEO were fabricated using DIW method and were assessed by a series of material characterisation. The type of solvent system had a noticeable impact on the ink rheology, which ultimately affected the surface properties. The incorporation of PEO particularly enhanced the roughness and wettability of the constructs. Our results support the use of DIW as a new means to process hydrophobic polymers for biomedical applications.

Electrospun zein nanofibers have attracted interest as drug delivery systems due to their propensity for controlled drug release, flexible structure and low toxicity. However, comparatively little is known regarding the relationship between production method and fiber characteristics, both in terms of fiber architecture and protein structure. Here we use a range of imaging and spectroscopic techniques to elucidate the effects of solvent composition on zein secondary structure, fiber diameter and fiber integrity, plus we utilize the new technique of transition temperature microscopy to examine the thermal properties of the fibers. Zein nanofibers were prepared using ethanol, acetic acid and water mixes as solvents, alone and with plasticizers (polyethylene glycol, glycerol) and casein. Electrospinning was performed under controlled conditions and the products characterized using scanning electron microscopy (SEM), attenuated total reflection Fourier Transform infrared spectrometry (ATR - FTIR) and transition temperature microscopy (TTM). The choice of solvent, concentration and voltage, alongside the presence of additives (plasticizers and casein) were noted to influence both the diameter of the fibers and the tendency for bead formation. A relationship was noted between protein secondary structure and fiber architecture, with an enhanced β-sheet content, enhanced by the inclusion of casein, being associated with higher beading. In addition, thermal imaging of electrospun zein fiber mats was successfully achieved using TTM via two dimensional mapping of the softening temperatures across the spun samples, in particular demonstrating the plasticizing effects of the polyethylene glycol and glycerol.

The orally disintegrating tablet (ODT) has shown vast potential as an alternative oral dosage form to conventional tablets wherein they can disintegrate rapidly (&le, 30 s) upon contact with saliva fluid and should have an acceptable mouthfeel as long as their weight doesn&rsquo, t exceed 500 mg. However, owing to the bitterness of several active ingredients, there is a need to find a suitable alternative to ODTs that maintains their features and can be taste-masked more simply and inexpensively. Therefore, electrospun nanofibers and solvent-cast oral dispersible films (ODFs) are used in this study as potential OD formulations for prednisolone sodium phosphate (PSP) that is commercially available as ODTs. The encapsulation efficiency (EE%) of the ODFs was higher (&asymp, 100%) compared to the nanofibers (&asymp, 87%), while the disintegration time was considerably faster for the electrospun nanofibers (&asymp, 30 s) than the solvent-cast ODFs (&asymp, 700 s). Hence, accelerated release rate of PSP from the nanofibers was obtained, due to their higher surface area and characteristic surface morphology that permitted higher wettability and thus, faster erosion. Taste-assessment study using the electronic-tongue quantified the bitterness threshold of the drug and its aversiveness concentration (2.79 mM). Therefore, a taste-masking strategy would be useful when further formulating PSP as an OD formulation.

Genetic transformation of tomato was first accomplished around 30 years ago. However, variability in transformation efficiency of distinct cultivars exists and to some extent remains a bottleneck for transgenic research. This study reports strategies to improve transformation efficiency in tomato and investigates regeneration capacity of transgenic plants under different selection regimes and hormonal applications. Tomato cv. Rio Grande was used as plant material and hygromycin and phosphinothricin (PPT) were used as selection agents. We found that cv. Rio Grande inherently produced a significant number of abnormal (“blind”) shoots lacking an apical meristem. Replacing cytokinin zeatin riboside with 6-benzylaminopurine (BAP) reduced the number of blind shoots although it slightly prolonged regeneration time. Survival rate of calli and shoots was very low using PPT as selection, whereas regeneration was achieved using hygromycin. Delayed application of hygromycin selection following co-cultivation with Agrobacterium tumefaciens improved the overall callus and shoot production. In vivo GFP fluorescence was detected to investigate the development of transgenic tissues using different hygromycin selection regimes. Higher transformation frequency was achieved when explants were continuously exposed to selection agents immediately following the pre-selection stage. Reducing the selection period followed by a non-selection stage increased the number of shoots, but these shoots were mostly non-transgenic. Thus, although less stringent selection, as expected, encouraged regeneration of shoots from calli, it did not improve transformation efficiency. Omitting selection altogether greatly reduced the efficiency of transformation. It was concluded that BAP is more suitable for normal shoot development, and that delayed selection followed by continuous selection results in higher transformation frequency. A significant improvement in regeneration of transgenic tomato through identification of key regeneration factors and robust screening is described, enabling high throughput genetic engineering in tomato.

Susannah Spurgeon (1832–1903) was married to the renowned London Baptist preacher, Charles Haddon Spurgeon (1834–1892). Fully supportive of his work, she organised a Book Fund which sent his books and sermons around the world to impoverished ministers. She often compared the growth of the fund to a lemon tree which she had planted and used stories about it to inspire giving to the fund. This article traces the growth of the fund, highlights the work of Susannah Spurgeon and suggests that much of C.H. Spurgeon’s world-wide fame may be traced to the efforts of what became known as Mrs C.H. Spurgeon’s Book Fund

Background. Compliance aids are devices which have been developed and are currently used to assist individuals in their medicines management. The use of compliance aids involves the transfer of medicines from the manufacturer’s original packaging and repackaged into an multi-compartment compliance aid (MCA). MCAs do not guarantee the same level of protection compared to manufacturer's original packaging. Objective. The aim of this study was to investigate the stability profile of atenolol, aspirin and lansoprazole dosage forms repackaged together in two differentcommercially available MCAs. Methods. In a laboratory in the United Kingdom, the physical stability of the formulations repackaged into two commercially available brands of MCAs was evaluated. After 8 weeks of storage (under controlled ambient conditions), changes in the disintegration (tablets only) and dissolution properties (all formulations) were examined in accordance with British Pharmacopoeia (BP) specifications. Key Findings. Findings from this study confirm that changes in solid dosage form quality are observed when repackaged into MCAs compared to manufacturers packaging resulting in differences in in vitro dissolution performance. However, even with these changes overall product performance was acceptable and within BP specifications. Conclusion. There is a need for greater collaboration in this area between manufacturers, hospital and community pharmacists, academics and policy makers to increase the data available on the physical stability and in turn performance of medicines repackaged into MCAs.

Virus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment. Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34Â nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the three-dimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids. HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids.

Corneal abrasion is a scratch wound on the surface of the anterior segment of the eye, which can predispose a patient to corneal infection and scarring, particularly if the cut penetrates to the deep corneal layers. Here we investigate a novel approach to co-administer an anti-scarring agent and an antibiotic, both being incorporated into one dosage form so as to accelerate wound closure and to treat any associated infection. More specifically, we have used electrospun fibers as a means of incorporating the two drugs into distinct compartments via coaxial electrospinning. Samples were characterised using a range of imaging, spectroscopic and thermal methods, while an HPLC assay has been developed to allow measurement of the concentration of both drug components in both the initial fibers and on release. Fibers loaded with pirfenidone in the hydrophobic polymer, PLGA, as the outer layer and moxifloxacin in the hydrophilic polymer PVP as the inner layer were successfully prepared, with smooth and non-porous surfaces and a mean diameter of circa 630 nm. TEM image demonstrated clear distinctive layers (a core and a shell), suggesting the successful preparation of the drug-loaded coaxial fibers, supported by HPLC entrapment studies, while fluorescence microscopy confirmed the presence of the moxifloxacin within the fibers. The fibers were capable of extending the release of both drugs, hence raising the possibility of a single daily dose of the drug-loaded coaxial fibers for the treatment of corneal abrasion.

The treatment of corneal abrasion currently involves the topical administration of antibiotics, with moxifloxacin HCl (0.5% w/v) eye drops being one of the most widely used treatments. Our previous work (Tawfik et al., 2020) involved the development of coaxial poly-lactic-co-glycolic acid (PLGA) and polyvinylpyrrolidone (PVP) nanofibers loaded with the antibiotic moxifloxacin HCl and the anti-scarring agent pirfenidone in the core (PVP) and shell (PLGA) respectively, with a view to the system comprising an ocular insert for the combination therapy of corneal abrasion. In this study, we examine the antimicrobial, anti-scarring and pharmacokinetic properties of the fibers alongside consideration of their toxicity and propensity for irritation. Minimum inhibitory concentration and zone of inhibition studies against S. aureus and P. aeruginosa were performed, while fibroblast cell viability and α-smooth muscle actin (α-SMA, a biomarker for scar formation) were measured using MTT and Western Blot assays, respectively. Pharmacokinetic studies and efficacy against infection were performed using a rabbit model, while ocular irritancy was assessed using the Draize test. The studies demonstrated that the antimicrobial activity of the moxifloxacin HCl was preserved following encapsulation into the nanofibers, while the downregulation of α-SMA was demonstrated using concentrations below the IC20 values (concentration required to decrease corneal fibroblast viability by no more than 20%). The pharmacokinetic study showed retention and sustained release of the moxifloxacin HCl over a 24-hour period, in contrast to equivalent eye drops which required four times daily dosing. Evidence of low level (according to the MMTS scale) irritation was detected for the nanofiber systems. Overall, the study has demonstrated that the dual drug-loaded nanofiber system shows potential for once daily dosing as an ocular insert for the treatment of corneal abrasion.

Narrow-leaf lupin (NLL) is the main legume crop grown in rotation with wheat and other cereals in Western Australia. Efforts to improve NLL germplasm by use of genetic technologies have been hampered by the lack of an efficient genetic transformation method, an issue that is in common with dominant crop legumes globally. Prior research has primarily used the bar gene for phosphinothricin (PPT) resistance. The aim of recent research has been to investigate alternative selection methodologies, in order to determine whether the limitations of low frequency of transgenic shoots, combined with chimerism at T0 could be overcome. Investigation of hygromycin resistance as a selectable marker compared to PPT is reported here. The results suggested that hygromycin resistance was a more suitable selectable marker for NLL transformation than PPT. Surprisingly, from investigation of transformation using the GUS reporter gene, it was also observed that transformation frequency was greater when selection treatment was reduced or delayed, compared to the existing protocols. To further investigate this observation, an eGFP expressing construct was prepared. Observations within the first week after Agrobacterium exposure of lupin explants demonstrated that transformation of NLL explant cells was not a rate-limiting step. Instead, the results indicated that the current selection methodology was killing the cells that were competent to regenerate into transgenic shoots. It was concluded that further research on the development of the treated explants should focus on delayed selection and exposure to Agrobacterium of cells below the apical meristem.

For pulse legume research to fully capitalise on developments in plant molecular genetics, a high throughput genetic transformation methodology is required. In Western Australia the dominant grain legume is Lupinus angustifolius L. (narrow leafed lupin; NLL). Standard transformation methodology utilising Agrobacterium tumefaciens on wounded NLL seedling shoot apices, in combination with two different herbicide selections (phosphinothricin and glyphosate) is time consuming, inefficient, and produces chimeric shoots that often fail to yield transgenic progeny. Investigation of hygromycin as an alternative selection in combination with expression of green fluorescent protein indicated that transformation of NLL apical cells was not the rate limiting step to achieve transgenic shoot materials. In this research it was identified that despite ready transformation, apical cells were not competent to regenerate. However a deep and broad wounding procedure to expose underlying axillary shoot and vascular cells to Agrobacterium, in combination with delayed selection proved successful, increasing initial explants transformation efficiency up to 75 % and generating axillary shoots with significant transgenic content. Based on knowledge gained from studies of plant chimeras, further subculture of these initial axillary shoots will result in development of low chimeric transgenic materials with heritable content. Furthermore, the method was also tested successfully on other Lupinus species, faba bea and field pea. These results demonstrate that development of a high yielding transformation methodology for pulse legume crops is achievable.

Weed management is a critical issue in the production of narrow-leaf lupin (NLL) (Lupinus angustifolius L.). The gene pool has limited herbicide tolerance, early vigour or short season productivity, hampering conventional breeding approaches. Previous transformation of NLL used the bar gene encoding tolerance to the herbicide glufosinate, for selection. However that methodology achieved

This study describes the preparation of free films of zein with and without acetylated high amylose maize starch (HAS) and their corresponding coated tablets as a novel approach to colonic drug delivery. We hypothesise that the embedding of a digestible starch component within the inert zein would allow the film to remain intact until the large intestine is reached. Free films of zein alone and starch/zein were prepared and characterised. SEM and AFM images of film surface showed that films were morphologically inhomogeneous, particularly at lower HAS/Zein ratios; however, nanothermal analysis data suggested that these differences in appearance within the same film are not compositional differences, i.e. the systems are not phase-separated. Moreover, FT-IR could detect no molecular interaction between the two polymers. Paracetamol tablets were coated with HAS/Zein aqueous based coatings of different compositions to a TWG of 20%. Drug release from zein alone and 1:5 HAS/Zein coated tablets under upper gastrointestinal conditions (pH 1.2, pH 6.8 with pepsin and pancreatin included) was very similar (for example approximately 12% and 14% of the drug was released, respectively, after 6 hours in a sequential in vitro test), suggesting that release in this region is limited and is not influenced by the presence of HAS in the ratio to zein under study. Studies using an in vitro colon model showed that under simulated colonic conditions, the drug release was significantly (p < 0.05) more rapid from 1:5 HAS/Zein, compared to the zein alone coating formulation. These data therefore support the potential use of zein-starch mixed films for colonic targeting purposes.

The reduced mycorrhizal colonization (rmc) tomato mutant is unable to form mycorrhiza and is more susceptible to Fusarium wilt compared with its wild-type isogenic line 76R. The rmc mutant has a chromosomal deletion affecting five genes, one of which is similar to CYCLOPS. Loss of this gene is responsible for non-mycorrhizality in rmc but not enhanced Fusarium wilt susceptibility. Here, we describe assessment of a second gene in the rmc deletion, designated Solyc08g075770 that is expressed in roots. Sequence analyses show that Solyc08g075770 encodes a small transmembrane protein with putative phosphorylation and glycosylation sites. It is predicted to be localized in the plasma membrane and may function in transmembrane ion transport and/or as a cell surface receptor. Complementation and knock-out strategies were used to test its function. Some putative CRISPR/Cas-9 knock-out transgenic events exhibited Fusarium wilt susceptibility like rmc and some putative complementation lines were 76R-like, suggesting that the tomato Solyc08g075770 functions in Fusarium wilt tolerance. This is the first study to demonstrate that Solyc08g075770 is the contributor to the Tfw locus, conferring tolerance to Fusarium wilt in 76R which was lost in rmc.

Many plant species adapted to P-impoverished soils, including jarrah (Eucalyptus marginata), develop toxicity symptoms when exposed to high doses of phosphate (Pi) and its analogs such as phosphite (Phi) and arsenate (AsV). The present study was undertaken to investigate the effects of fungal symbionts Scutellospora calospora, Scleroderma sp., and Austroboletus occidentalis on the response of jarrah to highly toxic pulses (1.5 mmol kg(-1) soil) of Pi, Phi, and AsV. S. calospora formed an arbuscular mycorrhizal (AM) symbiosis while both Scleroderma sp. and A. occidentalis established a non-colonizing symbiosis with jarrah plants. All these interactions significantly improved jarrah growth and Pi uptake under P-limiting conditions. The AM fungal colonization naturally declines in AM-eucalypt symbioses after 2-3 months; however, in the present study, the high Pi pulse inhibited the decline of AM fungal colonization in jarrah. Four weeks after exposure to the Pi pulse, plants inoculated with S. calospora had significantly lower toxicity symptoms compared to non-mycorrhizal (NM) plants, and all fungal treatments induced tolerance against Phi toxicity in jarrah. However, no tolerance was observed for AsV-treated plants even though all inoculated plants had significantly lower shoot As concentrations than the NM plants. The transcript profile of five jarrah high-affinity phosphate transporter (PHT1 family) genes in roots was not altered in response to any of the fungal species tested. Interestingly, plants exposed to high Pi supplies for 1 day did not have reduced transcript levels for any of the five PHT1 genes in roots, and transcript abundance of four PHT1 genes actually increased. It is therefore suggested that jarrah, and perhaps other P-sensitive perennial species, respond positively to Pi available in the soil solution through increasing rather than decreasing the expression of selected PHT1 genes. Furthermore, Scleroderma sp. can be considered as a fungus with dual functional capacity capable of forming both ectomycorrhizal and non-colonizing associations, where both pathways are always accompanied by evident growth and nutritional benefits.

Mycorrhizal symbiosis requires several common symbiosis genes including CYCLOPS/IPD3. The reduced mycorrhizal colonisation (rmc) tomato mutant has a deletion of five genes including CYCLOPS/IPD3, and rmc is more susceptible to Fusarium wilt than its wild-type parental line. This study investigated the genetic defects leading to both fungal interaction phenotypes and whether these were separable. Complementation was performed in rmc to test the requirement for CYCLOPS/IPD3 in mycorrhiza formation and Fusarium wilt tolerance. Promoter analysis via GFP expression in roots was conducted to determine the role of native regulatory elements in the proper functioning of CYCLOPS/IPD3. CYCLOPS/IPD3 regulated by its native promoter, but not a 2×35S promoter, restores mycorrhizal association in rmc. GFP regulated by the 2×35S promoter is not expressed in epidermal cells of roots, indicating that expression of CYCLOPS/IPD3 in these cells is required for colonisation by the fungi utilised in this research. CYCLOPS/IPD3 did not restore Fusarium wilt tolerance, however, showing that the genetic requirements for mycorrhizal association and Fusarium wilt tolerance are different. Our results confirm the expected role of CYCLOPS/IPD3 in mycorrhizal symbiosis and suggest that Fusarium tolerance is conferred by one of the other four genes affected by the deletion.

Root-fungal symbioses such as mycorrhizas and endophytes are key components of terrestrial ecosystems. Diverse in trophy habits (obligate, facultative or hemi-biotrophs) and symbiotic relations (from mutualism to parasitism), these associations also show great variability in their root colonization and nutritional strategies. Specialized interface structures such as arbuscules and Hartig nets are formed by certain associations while others are restricted to non-specialized intercellular or intracellular hyphae in roots. In either case, there are documented examples of active nutrient exchange, reinforcing the fact that specialized structures used to define specific mycorrhizal associations are not essential for reciprocal exchange of nutrients and plant growth promotion. In feremycorrhiza (withAustroboletus occidentalisand eucalypts), the fungal partner markedly enhances plant growth and nutrient acquisition without colonizing roots, emphasizing that a conventional focus on structural form of associations may have resulted in important functional components of rhizospheres being overlooked. In support of this viewpoint, mycobiome studies using the state-of-the-art DNA sequencing technologies have unearthed much more complexity in root-fungal relationships than those discovered using the traditional morphology-based approaches. In this review, we explore the existing literature and most recent findings surrounding structure, functioning, and ecology of root-fungal symbiosis, which highlight the fact that plant fitness can be altered by taxonomically/ecologically diverse fungal symbionts regardless of root colonization and interface specialization. Furthermore, transition from saprotrophy to biotrophy seems to be a common event that occurs in diverse fungal lineages (consisting of root endophytes, soil saprotrophs, wood decayers etc.), and which may be accompanied by development of specialized interface structures and/or mycorrhiza-like effects on plant growth and nutrition.

Temperature-controlled, solvent-free centrifugal spinning may be used as a means of rapid production of amorphous solid dispersions in the form of drug-loaded sucrose microfibers. However, due to the high content of amorphous sucrose in the formulations, such microfibers may be highly hygroscopic and unstable on storage. In this study, we explore both the effects of water uptake of the microfibers and the consequences of deliberate recrystallization for the associated dissolution profiles. The stability of sucrose microfibers loaded with three selected BCS class II model drugs (itraconazole (ITZ), olanzapine (OLZ), and piroxicam (PRX)) was investigated under four different relative humidity conditions (11, 33, 53, and 75% RH) at 25 °C for 8 months, particularly focusing on the effect of the highest level of moisture (75% RH) on the morphology, size, drug distribution, physical state, and dissolution performance of microfibers. While all samples were stable at 11% RH, at 33% RH the ITZ-sucrose system showed greater resistance against devitrification compared to the OLZ- and PRX-sucrose systems. For all three samples, the freshly prepared microfibers showed enhanced dissolution and supersaturation compared to the drug alone and physical mixes; surprisingly, the dissolution advantage was largely maintained or even enhanced (in the case of ITZ) following the moisture-induced recrystallization under 75% RH. Therefore, this study suggests that the moisture-induced recrystallization process may result in considerable dissolution enhancement compared to the drug alone, while overcoming the physical stability risks associated with the amorphous state.

• Most terrestrial plants form mutually beneficial symbioses with specific soil-borne fungi known as mycorrhiza. In a typical mycorrhizal association, fungal hyphae colonize plant roots, explore the soil beyond the rhizosphere and provide host plants with nutrients that might be chemically or physically inaccessible to root systems. • Here, we combined nutritional, radioisotopic ((33)P) and genetic approaches to describe a plant growth promoting symbiosis between the basidiomycete fungus Austroboletus occidentalis and jarrah (Eucalyptus marginata), which has quite different characteristics. • We show that the fungal partner does not colonize plant roots; hyphae are localized to the rhizosphere soil and vicinity and consequently do not transfer nutrients located beyond the rhizosphere. Transcript profiling of two high-affinity phosphate (Pi) transporter genes (EmPHT1;1 and EmPHT1;2) and hyphal-mediated (33)Pi uptake suggest that the Pi uptake shifts from an epidermal to a hyphal pathway in ectomycorrhizal plants (Scleroderma sp.), similar to arbuscular mycorrhizal symbioses, whereas A. occidentalis benefits its host indirectly. The enhanced rhizosphere carboxylates are linked to growth and nutritional benefits in the novel symbiosis. • This work is a starting point for detailed mechanistic studies on other basidiomycete-woody plant relationships, where a continuum between heterotrophic rhizosphere fungi and plant beneficial symbioses is likely to exist.

Aims: The purpose of this study was to prepare and evaluate ocular thermosetting gels containing sparfloxacin -HP-�≤ -CyD. The formulations based mainly onPluronic F127 alone or combined with other polymers. Study Design:Experimental study.

The aim of the study was to prepare molecular dispersions of a physically highly unstable amorphous drug, paracetamol (acetaminophen with a T(g) of ca. 25°C) via co-spray drying with a variety of polymers. Solid dispersions at a range of drug loadings (10-90%w/w) using hydroxypropyl methylcellulose/acetate succinate (HPMC/HPMC AS), polyvinylpyrrolidone (PVP) and copovidone were produced and characterised by modulated temperature differential scanning calorimetry (MTDSC), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). PVP-based polymers showed a greater tendency than the HPMC-based group to generate temperature-stable dispersions. In particular, copovidone (Plasdone® S-630) was found to be the most effective of the polymers studied and could formulate molecular dispersions at drug loadings up to and including 40%w/w. However, no evidence for direct drug-polymer interactions was found for such systems as a possible stabilising mechanism. The expected relationship of a higher T(g) of the polymer leading to greater stabilisation was not observed, while there was an inverse relationship between viscosity grade and amorphous phase generation. The study has therefore shown that temperature-stable amorphous dispersions of a low T(g) drug may be prepared by co-spray drying, particularly using PVP-based polymers.

Aims: The purpose of this in vitro and ex vivo study was to prepare and characterise ocular minitablets of piroxicam based on different polymeric matrices and to evaluate their potential to provide prolonged and controlled drug release to ocular tissues after surface administration. Study Design: Experimental study and ex-vivo study. Place and Duration of Study: School of Pharmacy, University of East Anglia, Norwich, Norfolk, UK, between July 2011 and March 2012. Methodology: A range of placebo minitablet formulations were prepared based on pharmaceutically-acceptable polymers of differing chemical and physical properties. These were evaluated using standard physical and visual imaging methods. A subset of placebo formulations was chosen to prepare medicated minitablets containing 5 %w/w piroxicam as a model drug. Three different in vitro methodologies were used to assess drug release from the minitablets. An ex vivo porcine ocular method was used to assess likely tissue distribution of the drug after surface ocular administration of the minitablets. Results: Minitablets were successfully produced from all formulations. The in vitro drug release profile was dependent on the chemistry of the polymer used, its hydration and swelling behaviour and to some extent, the methodology used for assessing the drug

The high concentrations of Mn, Fe and Al in acid soils during waterlogging impair root and shoot growth more severely in intolerant than tolerant wheat genotypes. This study aims to establish whether this difference in vegetative growth and survival during waterlogging (1) is verifiable across a range of tolerant/intolerant genotypes and acid soils, and (2) results in improved recovery after cessation of waterlogging and enhanced grain yield. Wheat genotypes contrasting in their tolerance to ion toxicities were grown in four acid soils until 63DAS and maturity, with a 42-day waterlogging treatment imposed at 21 DAS. The shoot Al, Mn and Fe concentrations increased by up to 5-, 3- and 9-fold respectively due to waterlogging in various soils. Compared to the intolerant lines, Al-, Mn- and Fe-tolerant genotypes maintained a relatively lower increase in shoot concentrations of Al (79 vs. 117%), Mn (90 vs. 101%) and Fe (171 vs. 252%) and demonstrated better waterlogging tolerance at the vegetative stage expressed in relative root (38% vs. 25%) and shoot (62% vs. 52%) growth. After cessation of waterlogging and the continued growth to maturity, tolerant genotypes maintained a relatively lower plant concentration of Al, Mn and Fe, but produced a higher above-ground biomass (74% vs. 56%) and most importantly demonstrated improved waterlogging tolerance (a relative grain yield of 78% vs. 54%) compared to intolerant genotypes. Maturity following waterlogging stress was delayed less in tolerant than intolerant genotypes (114 vs. 124%, respectively), which would reduce the potential yield loss where post-anthesis coincides with drought. The results confirm the validity of a novel approach of enhancing waterlogging tolerance of wheat genotypes grown in acid soil via increased tolerance to ion toxicities.

Zein films containing salicylic acid (SA) and acetyl salicylic acid (ASA) between 2 and 10% (initial zein weight basis) with or without glycerol were evaluated for structure, mechanical and dissolution properties. The random coils, α helices and β sheets mainly governed the secondary structure of zein, depending on glycerol and level of model molecules. Adding ASA resulted in an increase in α helices whereas β sheets increased at the expense of α helices when SA was used. Including SA or ASA decreased the tensile strength and the stiffness of films containing glycerol indicating the synergistic effect of SA and ASA. The strain at failure decreased with increasing content of SA but increased with increasing level of ASA. The dissolution properties were glycerol and drug dependent. ASA release in comparison to SA was quite low. The release was only observed above 10% ASA whereas it was detected in all films containing SA. The possible interactions between active components and proteins are discussed together with their implications on the physical properties of zein films.

Narrow-leafed lupin (NLL; Lupinus angustifolius) is a recently domesticated but anciently propagated crop with significant value in rotation with cereals in Mediterranean climates. However, several fungal pathogens, traditionally termed necrotrophs, severely affect broad-acre production and there is limited genetic resistance in the NLL germplasm pool. Symptoms of many of these diseases appear as localized areas of dead cells exhibiting markers of programmed cell death. Based on our previous research, we hypothesized that engineered expression of the baculovirus anti-apoptotic p35 gene might reduce symptoms of these diseases. Using Agrobacterium tumefaciens-mediated transformation of a cultivar highly susceptible to several pathogens, 14 independent NLL lines containing both the p35 and bar genes were obtained (p35-NLL). Integration and expression of the transgenes were confirmed by polymerase chain reaction (PCR), progeny testing, Southern blot, Northern blot and reverse transcriptase-PCR analyses. Fecundity and nodulation were not altered in these lines. Third or fourth generation p35-NLL lines were challenged with necrotrophic fungal pathogens (anthracnose in stem and leaf, and Pleiochaeta root rot and leaf brown spot) in controlled environment conditions. Several p35-NLL lines had significantly reduced disease symptoms. Interestingly, as with natural resistance, no single line was improved for all three diseases which possibly reflecting spatial variation of p35 expression in planta. These data support an alternative molecular definition for 'necrotrophic disease' in plants and suggest new routes for achieving resistance against a range of pathogens.

Kafirin and protein bodies were extracted from a condensed tannin-free white Sudanese cultivar of sorghum (Dabar). The extracted materials were characterized by SDS-PAGE. The potential of kafirin as a tablet matrix for pharmaceutical applications was studied. Tablets composed of kafirin, calcium hydrogen orthophosphate, caffeine and magnesium stearate were prepared by direct compression. The tablets showed appropriate levels of hardness and friability. Drug release studies showed that caffeine dissolution was greater in 0.1 M HCl than in either phosphate buffer (pH = 6.8) or distilled water. Deamidation of the protein in acid conditions might explain this observation. FTIR analysis showed that the secondary structure of kafirin was found to be mainly governed by α helices with some β sheets. Upon tabletting, there was a change in protein conformation, which was recovered upon dissolution irrespective of the dissolution media. This might be explained by the loss of protein coil to coil interaction during tabletting (possibly due to the diluting effect of calcium hydrogen orthophosphate). This was later recovered when tablets were dissolved due to the hydrophobic interactions between the kafirin proteins. In summary, this work has shown that kafirin has a potential for use as a tablet for drug delivery.

Micronutrient malnutrition, often called 'hidden hunger', affects over two billion people globally. This is particularly problematic in developing countries where widespread zinc (Zn) deficiency exists as a result of a predominantly plant-based diet. Furthermore, supplemental fertilizers are often unavailable or unaffordable in impoverished regions where soil infertility is common. Delivery of more Zn via food grains is theoretically possible through selective breeding strategies, but severe technical difficulties associated with trace element research have limited research on the underlying genetic components of Zn nutrition. Genetic dissection of Zn nutrition involved a pre-existing doubled haploid mapping population of barley (Hordeum vulgare). Association of mineral nutrient accumulation traits with regions of the barley genome was determined in two seasons of growth to maturity, using mapmanager qtx and QGene 4.0. Nine genetic loci segregating in the population associated clearly with measured traits, including five that contributed to grain Zn status. Pooling two-row doubled haploids by selecting the three most favourable alleles increased grain Zn content and concentration by an average of 53 and 75%, respectively. These results will inform breeding efforts for increased Zn density in the major food grain, wheat (Triticum aestivum), by enabling syntenic marker-assisted selection in conventional breeding programmes.

ObjectivesA liposome preparation that is amenable to receptor-mediated endocytosis has been developed to enhance the oral bioavailability of poorly absorbable peptidomi-metic drugs by use of folic acid as the mediator of liposomal uptake.MethodsFolic acid was physically coupled to the surface of the liposomes and cefotaxime was used as the model drug. In-vivo evaluation was carried out on eight Sprague-Dawley rats in a two-way crossover study to compare the oral bioavailability of cefotaxime loaded in folic acid-free liposomes and folic acid-coupled liposomes. Blood samples were collected from the tail vein and plasma cefotaxime levels were determined using an HPLC method.Key findingsEnhanced oral bioavailability (AUC0-∞) of cefotaxime was observed when administered via folic acid-coupled liposomes. The peak plasma concentration (Cmax) of cefotaxime was increased when administered via folic acid-coupled liposomes as compared with folic acid-free liposomes. At 90% confidence interval, the value for AUC0-∞ was 1.4–2-times higher and the value for Cmax was 1.2–1.8-times higher for the folic acid-coupled liposomes compared with folic acid-free liposomes.ConclusionsFolic acid could enhance the uptake of liposomally entrapped drug. It could be a useful candidate to supplement liposome delivery systems.

Zein, the main seed storage protein of maize, has been widely studied as a possible source of material for the production of biodegradable plastic films. Plasticization of zein is critical to make functional films. While there have been a number of publications which report the behavior of systems with a wide variety of plasticizers, there have been few which attempt to examine the interactions of protein and plasticizer at the molecular level. In this paper, we report on the plasticizing effects of water, glycerol, and 2-mercaptoethanol, which were examined by a combination of spectroscopy (FTIR and dielectric) and thermomechanical methods. The results suggest that both water and glycerol are adsorbed onto the protein and form hydrogen bonds with the amide groups. The plasticizer then builds up in patches on the protein surface. 2-Mercaptoethanol only exhibited a weak plasticizing effect due probably to disulfide bond breaking.

Members of the Australian native perennial Fabaceae have been little explored with regard to their root biology and the role played by arbuscular mycorrhizal (AM) fungi in their establishment, nutrition and long-term health. The ultimate goal of our research is to determine the dependency of native perennial legumes on their co-evolved AM fungi and conversely, the impact of AM fungal species in agricultural fields on the productivity of sown native perennial legume pastures. In this paper we investigate the colonisation morphology in roots and the AMF, identified by spores extracted from rhizosphere soil, from three replicate plots of each of the native legumes, Cullen australasicum, C. tenax and Lotus australis and the exotic legumes L. pedunculatus and Medicago sativa. The plants were grown in an agricultural field. The level and density of colonisation by AM fungi, and the frequency of intraradical and extraradical hyphae, arbuscules, intraradical spores and hyphal coils all differed between host plants and did not consistently differ between native and exotic species. However, there were strong similarities between species in the same genus. The three dominant species of AM fungi in rhizosphere soil also differed with host plant, but one fungus (Glomus mosseae) was always the most dominant. Sub-dominant AM species were the same between species in the same genus. No consistent differences in dominant spores were observed between the exotic and native Fabaceae species. Our results suggest that plant host influences the mycorrhizal community in the rhizosphere soil and that structural and functional differences in the symbiosis may occur at the plant genus level, not the species level or due to provenance.

Summary The foods of Great and Sooty Shearwaters Puffinus gravis and P. griseus are described from birds collected off eastern Canada. There was a broad overlap in diet, but Great Shearwaters tended to take more squid and tough-bodied fish such as mackerel Scomber scombrus while Sooties took more euphausiids Meganyctiphanes norvegica and soft-bodied fish such as Herring Clupea harengus. These differences are apparently related to differences in bill structure and in the degree of adaptation to underwater swimming. The birds appeared to feed opportunistically on whatever prey was locally available in the size-range between euphausiids and small fish and squid: Meganyctiphanes off southwest Nova Scotia, spawning and post-spawning capelin Mallotus villosus off eastern Newfoundland, and migrating squid Illex illecebrosus on the Grand Bank. Possible factors influencing prey selection are discussed. It is suggested that the timing and routes of the birds' migrations in the North Atlantic are related to the exploitation of such local concentrations. Despite the overlap in diets, differences in the distributions of the two species rule out the possibility of significant competition for common food resources.

The nature of the hydration of proteins and the subsequent implications for functionality is a matter of importance in both pharmaceutical and food applications. Most published studies rely on the use of one technique and attempt to characterize the system. Few studies have used combinations of techniques. In this paper we report on the use of infrared, dielectric, and calorimetric methods to examine the hydration process of wheat gluten. This has been the subject of considerable study by other techniques and has been well characterized by our group. Results show that in both the infrared and dielectric measurements there is a change in behavior at about 35% water content. This is also the water content below which lowering the temperature of the sample does not result in ice formation. We suggest that at this water content the protein amide groups are fully hydrated, and beyond this point addition of water results in protein dilution rather than further hydration.

Our research aims to investigate the molecular communication between land plants and arbuscular mycorrhizal (AM) fungi in the establishment of symbiosis. We have identified a mutation in the facultative AM host tomato, which we named rmc. Plants that are homozygous for rmc no longer host most AM fungi. The mutation also affects the interaction of tomato with root knot nematode and Fusarium wilt. However, the function/s encoded by the intact Rmc locus is/are unknown. To clone and sequence the gene or genes that comprise the Rmc locus, we have initiated a positional cloning project. In this paper, we report the construction of mapping populations and use of molecular markers from the published genome map to identify the location of Rmc on tomato chromosome 8. Nucleotide binding site-leucine rich repeat resistance genes, reported to reside in the same region of that chromosome, provided insufficient differences to develop cleaved amplified polymorphic sequence markers. Therefore, we were unable to map these sequences in relation to rmc. Our results potentiate future work to identify the Rmc function and to determine the genetic basis for the multiple plant-microbe interaction functions that the rmc mutation has defined.

We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.

Muon spin relaxation of two liposome samples consisting of 100% DPPC (dipalmitoylphosphatidylcholine) and 50:50 mole% DPPC:Cholesterol at a final lipid concentration of 100 mg/mL are compared. The observation of a significant loss in the muonium fraction from 11% to 6% when cholesterol is present is compatible with radical formation in the second sample. Also reported are measurements on pure solid cholesterol where a clear T1 minimum at ca 100 K, corresponding to a dynamic process with activation energy of ca. 1.89 kJ mol−1 and an attempt frequency of 5.89×1010 s−1, is observed. An estimate of a hyperfine interaction of 255.4 MHz for this radical predicts a ΔM=1 ALC transition at 0.94 T.

The reduced arbuscular mycorrhizal (AM) colonization mutant of tomato (rmc) is a single locus recessive mutation with different colonization phenotypes for different AM fungi. To test broader specificity and gain possible functional insights, we compared host status of rmc and near isogenic parent (Rio Grande 76R) for three root parasites. No significant colonization differences were found for bulb-and-potato aphid. However, root knot nematode (RKN) grew significantly better on rmc root cultures. Also, rmc was significantly more susceptible than 76R to Fusarium wilt. Our results indicate that the Rmc locus may have dual roles in symbiosis and parasitism.

Self incompatibility (SI) in Phalaris coerulescens is gametophytically determined by two unlinked multi allelic loci (S and Z). Neither the S nor Z genes have yet been cloned. As part of a map-based cloning strategy, high-resolution maps of the S and Z regions were generated from distorted segregating populations using RFLP probes from wheat, barley, oat, and Phalaris. The S locus was delimited to 0.26 cM with two boundary markers (Xwg811 and Xpsr168) and cosegregated with Xbm2 and Xbcd762. Xbcd266 was the closest marker linked to Z (0.9 cM). A high level of colinearity in the S and Z regions was found in both self-incompatible and -compatible species. The S locus was localized to the subcentromere region of chromosome 1 and the Z locus to the long arm end of chromosome 2. Several rice BAC clones orthologous to the S and Z locus regions were identified. This opens the possibility of using the rice genome sequence data to generate more closely linked markers and identify SI candidate genes. These results add further support to the conservation of gene order in the S and Z regions of the grass genomes.Key words: Phalaris coerulescens, self-incompatibility, distorted segregation, mapping, map-based cloning, synteny mapping.

A high-performance liquid chromatographic method with ultraviolet (UV) detection was developed for measuring cefotaxime in rat and human plasma. The method used direct injection of the plasma supernatant after deproteinization with 70% perchloric acid. Degradation of cefotaxime in acidic medium was retarded by adding phosphate buffer before centrifuging the sample. The mobile phase was 0.05 M aqueous ammonium acetate-acetonitrile-tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5. Analysis was run at a flow-rate of 1.0 ml/min, and a detection wavelength of 254 nm was used. The method has a quantification limit of 0.20 microgram/ml. The within- and between-day coefficients of variation and accuracy values were less than 8% and +/-3%, respectively, while the recovery values were greater than 87% over the concentration range tested (0.20-50 microgram/ml). The speed, sensitivity, specificity and reproducibility of this method make it particularly suitable for the routine determination of cefotaxime in human plasma. Moreover, only a relatively small sample plasma volume (100 microliter) is required, allowing this method to be applied to samples taken from neonates.

Genetic analyses of mycorrhizal symbioses have been far less common to date than molecular biological investigations. This review aims to address the problem that genetic research approaches are some of the least familiar to non specialists by providing some detailed explanations of the requirements and processes involved, including concepts of genetic variation and genetic mapping. Each section includes examples of research progress which is restricted to studies of arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) symbioses. Most such research has focussed on AM hosts or EcM fungi. For AM hosts, some early work on natural genetic variation has not been exploited yet, but new research with barley and clover will enable genetic mapping of mycorrhizal associated QTLs for the first time. EcM fungal studies have shown a genetic basis for mycorrhizal capacity and quantitative genetic differences in mycorrhizal capacity. Some recent work with EcM hosts has begun genetic mapping of QTLs associated with mycorrhizal status. Most AM genetic research has focussed on analysis of nodulation-defective mutants for their AM host status. Map-based cloning and characterisation of the first genes shown by these analyses to be essential for establishment of both nodulation and mycorrhizal symbioses are anticipated shortly. Comparisons with molecular and genetic research on plant disease resistance genes and signalling pathways may prove useful as those studies are more advanced and underlying biochemical and evolutionary relationships are likely to exist.

Trehalose dihydrate is a nonreducing disaccharide which has generated great interest in the food and pharmaceutical industries. However, it is well recognized that considerable batch to batch variation exists for supposedly identical samples, particularly in terms of the thermal response. In this investigation, two standardized forms of trehalose dihydrate were generated using two distinct crystallization pathways. The two batches were characterized using scanning electron microscopy, X-ray powder diffraction, and FTIR. The thermal responses of the two forms were then studied using modulated temperature differential scanning calorimetry (MTDSC) and thermogravimetric analysis (TGA). In particular, we describe the technique of quasi-isothermal MTDSC as a means of studying the change in equilibrium heat capacity as a function of temperature. Finally, variable temperature FTIR was utilized to assess the change in bonding configuration as a function of temperature. SEM revealed significant differences in the continuity and grain structure of the two batches. The TGA, MTDSC, and quasi-isothermal MTDSC studies all indicated significant differences in the thermal response and water loss profile. This was confirmed using variable temperature FTIR which indicated differences in bond reconfiguration as a function of temperature. We ascribe these differences to variations in the route by which water may leave the structure, possibly associated with grain size. The study has therefore demonstrated that chemically identical dihydrate forms may show significant differences in thermal response. We believe that this may assist in interpreting and hence controlling interbatch variation for this material.

Using linkage analysis, we identified a novel dominant locus, DFNA25, for delayed-onset, progressive, high-frequency, nonsyndromic sensorineural hearing loss in a large, multigenerational United States family of Czech descent. On the basis of recombinations in affected individuals, we determined that DFNA25 is located in a 20-cM region of chromosome 12q21-24 between D12S327 (centromeric) and D12S84 (telomeric), with a maximum two-point LOD score of 6.82, at recombination fraction .041, for D12S1030. Candidate genes in this region include ATP2A2, ATP2B1, UBE3B, and VR-OAC. DFNA25 may be the human ortholog of bronx waltzer (bv).

A molecular study of the mycorrhizal symbiosis between barley and Glomus intraradices used differential display PCR and a synchronous colonization method to identify genes that are differentially expressed in symbiosis. Several PCR products were consistently differentially amplified. PCR amplification of genomic DNA from either G. intraradices or barley as templates showed that three such products were encoded by G. intraradices. Sequence analysis of the deduced amino acid sequences of the fungal fragments, following extension by 3’-RACE, revealed similarities to proteins from higher eukaryotes. One (GINMYC1) shows similarity to TRIP15, a human protein that interacts in a hormone-dependent manner with the thyroid receptor. A second (GINMYC2) is similar to O-linked N-acetylglucosamine transferases from vertebrates, and the third (GINHB1) contains a putative leucine zipper and a homeodomain which indicates that it binds DNA and may act as a transcriptional regulator. Fragments of the expected sizes were amplified by RT-PCR from mRNA of mycorrhizal barley roots for all three fungal cDNAs, which indicates that the corresponding genes are expressed during intraradical growth of G. intraradices. The results provide a promising insight to fungal gene expression early in formation of this compatible and mutualistic symbiosis.