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Fruit growth and development are physiological processes controlled by several internal and external factors. This complex regulatory mechanism comprises a series of events occurring in a chronological order over a growing season. Understanding the underlying mechanism of fruit development events, however, requires consideration of the events occurring prior to fruit development such as flowering, pollination, fertilization, and fruit set. Such events are interrelated and occur in a sequential order. Recent advances in high-throughput sequencing technology in conjunction with improved statistical and computational methods have empowered science to identify some of the major molecular components and mechanisms involved in the regulation of fruit growth and have supplied encouraging successes in associating genotypic differentiation with phenotypic observations. As a result, multiple approaches have been developed to dissect such complex regulatory machinery and understand the genetic basis controlling these processes. These methods include transcriptomic analysis, quantitative trait loci (QTLs) mapping, whole-genome approach, and epigenetics analyses. This review offers a comprehensive overview of the molecular, genomic and epigenetics perspective of apple fruit growth and development that defines the final fruit size and provides a detailed analysis of the mechanisms by which fruit growth and development are controlled. Though the main emphasis of this article is on the molecular, genomic and epigenetics aspects of fruit growth and development, we will also deliver a brief overview on events occurring prior to fruit growth.

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Multi-scale models can facilitate whole plant simulations by linking gene networks, protein synthesis, metabolic pathways, physiology, and growth. Whole plant models can be further integrated with ecosystem, weather, and climate models to predict how various interactions respond to environmental perturbations. These models have the potential to fill in missing mechanistic details and generate new hypotheses to prioritize directed engineering efforts. Outcomes will potentially accelerate improvement of crop yield, sustainability, and increase future food security. It is time for a paradigm shift in plant modeling, from largely isolated efforts to a connected community that takes advantage of advances in high performance computing and mechanistic understanding of plant processes. Tools for guiding future crop breeding and engineering, understanding the implications of discoveries at the molecular level for whole plant behavior, and improved prediction of plant and ecosystem responses to the environment are urgently needed. The purpose of this perspective is to introduce Crops in silico (cropsinsilico.org), an integrative and multi-scale modeling platform, as one solution that combines isolated modeling efforts toward the generation of virtual crops, which is open and accessible to the entire plant biology community. The major challenges involved both in the development and deployment of a shared, multi-scale modeling platform, which are summarized in this prospectus, were recently identified during the first Crops in silico Symposium and Workshop.

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Fruit size regulation was studied in the apple cultivar 'Gala' and a large fruit size spontaneous mutant of 'Gala', 'Grand Gala' (GG). GG fruits were 15% larger in diameter and 38% heavier than 'Gala' fruits, largely due to an increase in size of the fruit cortex. The mutation in GG altered growth prior to fruit set and during fruit development. Prior to fruit set, the carpel/floral-tube size was enhanced in GG and was associated with higher cell number, larger cell size, and increased ploidy through endoreduplication, an altered form of the cell cycle normally absent in apple. The data suggest that the mutation in GG promotes either cell production or endoreduplication in the carpel/floral-tube cells depending on their competence for division. Ploidy was not altered in GG leaves. During fruit growth, GG fruit cells exited cell production earlier, and with a DNA content of 4C suggesting G2 arrest. Cell size was higher in GG fruits during exit from cell production and at later stages of fruit growth. Final cell diameter in GG fruit cortex cells was 15% higher than that in 'Gala' indicating that enhanced fruit size in GG was facilitated by increased cell size. The normal progression of cell expansion in cells arrested in G2 may account for the increase in cell size. Quantitative RT-PCR analysis indicated higher MdCDKA1 expression and reduced MdCYCA2 expression during early fruit development in GG fruits. Together, the data indicate an important role for cell expansion in regulating apple fruit size.

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PURPOSE: To compare the pulmonary pharmacokinetics and relative bioavailability of salmon calcitonin delivered as aqueous droplets, pH 6.6 and pH 4.8 with that of a spray dried powder in healthy volunteers. METHODS: Spray dried powders (1.6 microm [GSD 2.1]) containing 5% by wt. sCal, 6.25% human serum albumin, 73.55% mannitol and 15% citric acid/sodium citrate were prepared using a Buchi model 190 spray drier. Aqueous solutions were prepared by dissolving the spray dried powder at a sCal concentration of 1.25 mg/ml, pH was adjusted using 21 mM sodium hydroxide. Aerosols were delivered as part of a 4 way cross-over study to 16 healthy volunteers. The Nektar pulmonary delivery device was used to deliver the dry powder aerosol. A Salter nebulizer controlled by a Rosenthal dosimeter was used to deliver the aqueous aerosols. Miacalcin injection was used as the subcutaneous control. Dose delivered to the lung was estimated by gamma scintigraphy. Plasma concentrations of sCal were measured using a radioimmunoassay. RESULTS: Aerosol size distributions were matched, 3.3 microm MMAD and approximately 2.2 GSD. Inhaled flow rates were similar, although not equal, 5.8 and approximately 9.8 l/min respectively for dry powder and liquid inhalations. Lung doses of sCal ranged from 53 to 88 microgm, peripheral lung doses from 25 to 51 microgm. Pharmacokinetic profiles and lung bioavailability relative to subcutaneous injection for all formulations were similar (not statistically significantly different p > 0.05), relative lung bioavailability ranged from 11% to 18%, estimates of relative bioavailability based on peripheral lung dose ranged from 20% to 33%. CONCLUSION: The study showed no difference in pharmacokinetic profiles between the various aerosol dosage forms. pH of the aqueous solutions did not affect kinetics or relative bioavailability.

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STUDY OBJECTIVES: To evaluate the efficiency and reproducibility of pulmonary delivery of an investigational tobramycin PulmoSphere formulation (PStob) [Inhale Therapeutic Systems; San Carlos, CA] by a passive dry powder inhaler, and to compare serum concentrations and whole-lung deposition with a commercial nebulized tobramycin product (TOBI; Chiron Corporation; Seattle, WA). DESIGN: A five-period, open-label, nonrandomized crossover study. PARTICIPANTS: Fourteen healthy volunteers were studied, and 12 completed the study. INTERVENTIONS: PStob powder was manufactured using lipid-based PulmoSphere technology, producing highly dispersible porous particles. PStob was radiolabeled with (99m)Tc, and in vitro experiments confirmed it as a valid drug marker. To identify whole-lung distribution via scintigraphy, subjects inhaled contents of a single capsule (72 L/min) containing 25 mg of (99m)Tc-labeled PStob (13.5 mg of tobramycin free base) in periods 1 to 3. In period 4, subjects received (99m)Tc nebulized tobramycin, approximately 2.5 mL of 300 mg/5 mL. Deposition and blood samples were obtained. In period 5, six 25-mg doses of unlabeled PStob (81 mg of tobramycin base) were inhaled and blood samples were collected. MEASUREMENTS AND RESULTS: Mean whole-lung deposition of PStob was 34 +/- 6% and nebulized tobramycin was 5 +/- 2%. Peak tobramycin concentration in serum (Cmax) values were 0.6 microg/mL with PStob and 0.28 microg/mL after nebulized tobramycin. Serum area under the curve was 4.4 microg x h/mL vs 2.1 micro g x h/mL for nebulized tobramycin. Median time to Cmax for PStob was comparable to nebulized tobramycin. CONCLUSIONS: The aerosol doses of PStob (25 mg and 150 mg) were well dispersed and tolerated. Serum drug concentrations matched scintigraphy data and were roughly twice that of the comparator. Intrasubject dose variability for three equivalent periods did not exceed 18% relative SD. PStob Cmax (0.6 microg/mL) was well below the toxic threshold (2 micro g/mL).

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Whole lung and regional lung deposition of inhaled asthma drugs in the lungs can be quantified using either two-dimensional or three-dimensional radionuclide imaging methods. The two-dimensional method of gamma scintigraphy has been the most widely used, and is currently considered the industry standard, but the three-dimensional methods (SPECT, single photon emission computed tomography; and PET, positron emission tomography) give superior regional lung deposition data and will undoubtedly be used more frequently in future. Recent developments in radionuclide imaging are described, including an improved algorithm for assessing regional lung deposition in gamma scintigraphy, and a patent-protected radiolabelling method (TechneCoat), applicable to both gamma scintigraphy and SPECT. Radionuclide imaging data on new inhaled asthma products provide a milestone assessment, and the data form a bridge between in vitro testing and a full clinical trials program, allowing the latter to be entered with increased confidence.

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Dry powder inhalers (DPIs) are used to deliver asthma drugs to patients, but lung deposition may depend upon the degree of inspiratory effort. The pulmonary deposition of the glucocorticosteroid budesonide (SMB-Galephar) has been assessed in 12 asthmatic patients when delivered by the Monodose inhaler (Miat, Milan, Italy); the Pulmicort Turbuhaler DPI (AstraZeneca, Lund, Sweden) was used as a comparator product. Patients inhaled from each device with maximal or sub-maximal inspiratory effort: Monodose inhaler 90 vs 45 l/min; Turbuhaler DPI 60 vs 30 l/min. The formulations were radiolabelled with (99m)Tc, and deposition of budesonide was quantified by gamma scintigraphy. Mean (SD) whole lung deposition for the Monodose inhaler (% capsule dose), was independent of inspiratory effort (maximal: 21.4 (4.3)%; sub-maximal: 21.4 (7.5)%), while lung deposition for the Turbuhaler DPI (% metered dose) fell significantly with decreasing inspiratory effort (maximal: 25.1 (6.1)%; sub-maximal: 18.5 (6.5)%; P<0.05). The plasma concentrations of budesonide showed the same trends as the whole lung deposition data. The Monodose inhaler showed inspiratory effort-independent drug delivery characteristics, and could prove be a valuable low-cost alternative to more complex devices such as the Turbuhaler DPI. The Monodose inhaler may be especially useful in groups of patients unable to inhale maximally through DPIs, including young children and adult patients with severe respiratory impairment.

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PURPOSE: To assess the pulmonary deposition and pharmacokinetics of an engineered PulmoSphere powder relative to standard micronized drug when delivered from passive dry powder inhalers (DPIs). METHODS: Budesonide PulmoSphere (PSbud) powder was manufactured using an emulsion-based spray-drying process. Eight healthy subjects completed 3 treatments in crossover fashion: 370 microg budesonide PulmoSphere inhaled from Eclipse DPI at target PIF of 25 L x min(-1) (PSbud25), and 50 L x min(-1) (PSbud50), and 800 microg of pelletized budesonide from Pulmicort Turbuhaler at 60 L x min(-1)(THbud60). PSbud powder was radiolabeled with 99mTc and lung deposition determined scintigraphically. Plasma budesonide concentrations were measured for 12 h after inhalation. RESULTS: Pulmonary deposition (mean +/- sd) of PSbud was 57+/-7% and 58+/-8% of the nominal dose at 25 and 50 L x min(-1), respectively. Mean peak plasma budesonide levels were 4.7 (PSbud25), 4.0 (PSbud50), and 2.2 ng x ml(-1) (THbud60). Median t(max) was 5 min after both PSbud inhalations compared to 20 min for Turbuhaler (P < 0.05). Mean AUCs were comparable after all inhalations, 5.1 (PSbud25), 5.9 (PSbud50), and 6.0 (THbud60) ng x h x ml(-1). The engineered PSbud powder delivered at both flow rates from the Eclipse DPI was twice as efficiently deposited as pelletized budesonide delivered at 60 L x min(-1) from the Turbuhaler. Intersubject variability was also dramatically decreased for PSbud relative to THbud. CONCLUSION: Delivery of an engineered PulmoSphere formulation is more efficient and reproducible than delivery of micronized drug from passive DPIs.

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PURPOSE: PulmoSphere particles are specifically engineered for delivery by the pulmonary route with a hollow and porous morphology, physical diameters < 5 microm, and low tap densities (circa 0.1 g x cm(-3)). Deposition of PulmoSphere particles in the human respiratory tract delivered by pressurized metered dose inhaler (pMDI) was compared with deposition of a conventional micronized drug pMDI formulation. METHODS: Nine healthy nonsmoking subjects (5 male, 4 female) completed a two-way crossover gamma scintigraphic study, assessing the lung and oropharyngeal depositions of albuterol sulfate, formulated as 99mTc-radiolabeled PulmoSphere particles or micronized particles (Ventolin Evohaler, GlaxoSmithKline, Ltd.) suspended in HFA-134a propellant. RESULTS: Mean (standard deviation) lung deposition, (% ex-valve dose) was doubled for the PulmoSphere formulation compared with Evohaler pMDI (28.5 (11.3) % vs. 14.5 (8.1) %, P < 0.01), whereas oropharyngeal deposition was reduced (42.6 (9.0) % vs. 72.0 (8.0) %, P < 0.01). Both PulmoSphere and Evohaler pMDIs gave uniform deposition patterns within the lungs. CONCLUSIONS: These data provided "proof of concept" in vivo for the PulmoSphere technology as a method of improving targeting of drugs to the lower respiratory tract from pMDIs, and suggested that the PulmoSphere technology may also be suitable for the delivery of systemically acting molecules absorbed via the lung.

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