超微电极实时监测植物细胞壁参与调控的单细胞活性氧爆发

植物细胞活性氧爆发在植物的抗病以及信号转导中起着非常重要的作用,植物内活性氧产生及代谢受到复杂而精确的机制调控,从而维持正常的活性氧水平以发挥其生理功能. 然而,在单细胞水平开展活性氧爆发实时监测及其调控机制研究一直受到很大的挑战. 本文以碳纤维微盘电极（CFMDE）为基底电极,利用Nafion的模板效应,采用电化学沉积法制得纳米铂颗粒修饰电极（NPt/Nafion/ CFMDE）;同时采用基于聚二甲基硅氧烷（PDMS）的软光刻技术,制备了一种高效固定植物悬浮细胞的琼脂糖阵列微孔芯片. 使用NPt/Nafion/CFMDE实时监测了单个拟南芥原生质体活性氧爆发,并证明电化学监测活性氧的主要成分为过氧化氢. 在此基础上,采用浅层培养法培养原生质体再生植物细胞壁. 电化学监测结果表明,与单个原生质体相比,植物细胞在受到刺激时释放的过氧化氢量显著降低;然而当采用过氧化物酶抑制剂抑制植物细胞壁上过氧化物酶活性后,植物细胞释放过氧化氢量显著回升. 研究结果表明细胞壁在活性氧爆发过程具有很好的调控功能,可望促进植物细胞活性氧爆发及其调控机制的研究.     

A “Polymer Template” Strategy for Carbonized Polymer Dots with Controllable Properties

Limited avenues are available for property control of carbonized polymer dots (PDs) owing to the unsatisfactory understanding of PDs“ formation. Herein, a de novo ”polymer template“ strategy is presented for PDs with customizable functional surface groups (FSG), size, and underlying fluorescence, with a detailed mechanism. The strategy relies on novel di-active site polymers (DASPs) prepared from alkenyl azides via [3+2] cycloaddition and guanidino hydrolysis. Benefiting from these specific reactions, the DASPs were convenient for mass production and stable for storage, and could be transformed to PDs upon addition of nucleophilic agents through nucleophilic addition and substitution at 70 °C. By regulating the types of alkenyl azides, nucleophilic agents, and reaction conditions, the as-prepare PDs could be tailored with controlled types of core, FSG, and particle size, as well as fluorescence properties of quantum yield from 8.2–55.6 %, and emission maximum from 380–500 nm. These specialties make this ”polymer template“ strategy a promising start for building PDs-based sensor platforms. Moreover, the strategy could further our understanding towards PDs’ formation, and open up a new way to customize PDs for specific needs in the fields of analysis, catalysis, images, etc.     

Introductory remarks

Separate terms to distinguish the matrix between the cells that grow in vitro and the matrix that separates and surrounds cells in the living body are being proposed. The currently used designations: pericellular, extracellular, or intercellular matrix should be used only for the substance produced by cells in vitro (cultures). The well organized, highly specific, and stable structure with significant topographic variations between the cells of living tissues and organs should be called biological matrix or briefly as biomatrix. My experiments published in the early 1940s on plant cell walls illustrate some similarities between biomatrix of animal tissues and plant cell wall. When cells of plant epithelial membranes undergo plasmolysis, the space between the contracting cell and cellulose cell wall is filled with filaments called Hechtian filaments or strands. With silver impregnation, these filaments become a fibrous network, filling the space between the cell wall and the contracting plasma (cell) membrane. A similar extracellular fibrous network has been described earlier after silver impregnation in some animal tissues. I interpreted my finding that a pectin-like polysaccharide in the cell wall biomatrix and hyaluronan, the only polysaccharide present in the biomatrix of animal tissues, are responsible for this fibrous network artifact.     

Template‐Synthesized Vertical Needle Array as Injection Platform for Microalgae

Increasing numbers of studies in the past few decades have demonstrated vertically‐oriented nanoneedles arrays (NNAs) as innovative tools to interrogate and manipulate biological cells, where the needles are inserted into the cells as functional probes for high‐throughput detection and biomolecule delivery. However, majority of these studies use mammalian cells: leaving NNA application to plant cells still in its infancy and largely unexplored. This paper highlights our contributions in exploring the utility of NNAs to microalgae – a diverse group of aquatic, photosynthetic organisms studied intensively as bio‐factories for producing high‐value‐added products such as fuels and pharmaceuticals. Microalgal strain development processes have long suffered from the hard cell wall that surrounds the cell and inhibits delivery of foreign materials into the cell. Conically‐shaped, metallic NNAs were developed with template synthesis that successfully penetrate through the cell wall barrier and achieve material injection – using the widely studied model microalga, Chlamydomonas reinhardtii. Earlier works from mammalian cells are introduced and discussed to clarify the framework established in this field, while recent studies of both mammalian and microalgal cells are also referenced to examine the trends, challenges, and future perspectives of NNA application to microalgae.     

Characterization of Natural and Alkaline-Oxidized Proanthocyanidins in Plant Extracts by Ultrahigh-Resolution UHPLC-MS/MS

In this study, we analyzed the proanthocyanidin (PA) composition of 55 plant extracts before and after alkaline oxidation by ultrahigh-resolution UHPLC-MS/MS. We characterized the natural PA structures in detail and studied the sophisticated changes in the modified PA structures and the typical patterns and models of reactions within different PA classes due to the oxidation. The natural PAs were A- and B-type PCs, PDs and PC/PD mixtures. In addition, we detected galloylated PAs. B-type PCs in different plant extracts were rather stable and showed no or minor modification due to the alkaline oxidation. For some samples, we detected the intramolecular reactions of PCs producing A-type ether linkages. A-type PCs were also rather stable with no or minor modification, but in some plants, the formation of additional ether linkages was detected. PAs containing PD units were more reactive. After alkaline oxidation, these PAs or their oxidation products were no longer detected by MS even though a different type and/or delayed PA hump was still detected by UV at 280 nm. Galloylated PAs were rather stable under alkaline oxidation if they were PC-based, but we detected the intramolecular conversion from B-type to A-type. Galloylated PDs were more reactive and reacted similarly to nongalloylated PDs.     

STUDY ON WAYS OF FORCING INTERCELLULAR CYTOPLASM AND CHROMATIN MIGRATION TO TRANSFER EXTERNAL GENETIC SUBSTANCES TO PLANT CELLS BY ARTIFICIAL MOTIVE FORCE

A new cell engineering technique (L. B. technique) was established in our Lab. At first, the physical and chemical methods were used to facilitate the reestablishment of intercellular contacts and plasmodesma channels between different parents, thus forcing cytoplasm and chromatin to pass the cell wall with different qualities and quantities from one cell to others, through the enlarged intercellular plasmodesma channels or the vulnerable regions and the holes on the cell wall formed differently in growth and thickness in the process of cell wall formation to introduce external genetic substances or gene groups into plant cells. There are different ways, frequencies and strengths for the migration between the cells in different growth and development regions or the same growth and development region. In this paper we advance the mechanism of cytoplasm and chromatin migration through the cell wall: There are a large number of plasmodesma channels or vulnerable regions and holes different in growth and th     

L'hydrogene pour le transport sur route : réalisations et développements

Hydrogen for road transportation : achievements and developments. At the beginning of this millenium, hydrogen appears as a potential energy carrier for the future. Thus, it could serve as a storage medium for renewable energy forms, which should play an increasing part in the world energy supply. In a closer future, hydrogen could also become a fuel for prospective fuel-cell and internal-combustion vehicles. We present here an inventory of the various technologies related to the use of hydrogen in road transportation : propulsion type (fuel cell and electric motor, or internal combustion engine), hydrogen production, on-board storage, infrastructure. Safety, standardization and regulation aspects will also be addressed. Presently, the majority of hydrogen buses are equipped with polymer membrane fuel cells (PEMFC), directly supplied with hydrogen from pressurized vessels (300 bars). On the other hand, car manufacturers are developing various types of experimental vehicles : internal-combustion engine cars with liquid hydrogen storage, fuel cell (PEMFC) cars with storage of hydrogen (liquid, gaseous, hydride) or of methanol. The type of required infrastructured will depend on the type of fuel chosen by the car makers and on the requirements of the oil companies. Several hydrogen supply stations, of different technologies, have already been set up. They deliver gaseous or liquid hydrogen produced by reforming of natural gas or by electrolysis. The building of a hydrogen-based fueling system requires the development of specific means of production, transportation, storage and delivery. Public acceptance will have to be won by guaranteeing safety, reliability, performance and competitivity. Presently, research and development work is mainly carried out on : on-board storage of hydrogen ; on-board systems for the production of hydrogen from methanol and petrol ; standardization and regulation.     

ICG and Sunitinib-loaded NH2-MOFs for Folate-mediated Hepatocellular Carcinoma Dual-modal Therapy

This research investigated a novel folic acid(FA)-modified zirconium core metal-organic framework(MOF) Uio-66 as a nanocarrier to deliver indocyanine green(ICG) and Sunitinib to cancer cells for combination therapy. Platinum-loaded Uio-66 nanoparticles(Pu) were synthesized via a one-pot method, followed by the modification with FA on their surfaces. This afforded FPu that enabled subsequent loading of ICG and Sunitinib to achieve dual-modal cancer therapy. Drug loading/release test and singlet oxygen detection were also conducted in vitro, and the nanoparticles showed considerable drug loading efficiency for both ICG and Sunitinib, coupled with a high singlet oxygen generation rate. Specifically, drug loading and encapsulation efficiency of Sunitinib were 2.30% and 72.67%, while those for ICG were 2.87% and 90.28%, respectively. Additionally, cytotoxicity test on HepG2 human hepatocellular carcinoma cancer cell line revealed that the fully functional nanoparticles possess excellent biocompatibility and as such could be further investigated as a potential drug delivery system for effectual carcinoma cancer treatment.     

Injectable Graphene Oxide/Graphene Composite Supramolecular Hydrogel for Delivery of Anti-Cancer Drugs

Injectable hydrogels have attracted a lot of attention in drug delivery, however, their capacity to deliver water-insoluble or hydrophobic anti-cancer drugs is limited. Here, we developed injectable graphene oxide/graphene composite supramolecular hydrogels to deliver anti-cancer drugs. Pluronic F-127 was used to stabilize graphene oxide (GO) and reduced graphene oxide (RGO) in solution, which was mixed with α-cyclodextrin (α-CD) solution to form hydrogels. Native hydrogel was used as control. GO or RGO slightly shortened gelation time. The storage and loss moduli of the hydrogels were tracked by dynamic force measurement. The storage modulus of GO or RGO composite hydrogels was larger than that of the native hydrogel. Hydrogels were unstable in solution and eroded gradually. GO or RGO in Pluronic F-127 solution could potentially improve the solubility of the water-insoluble anti-cancer drug camptothecin (CPT), especially with large drug-loaded CPT amount. Drug release behaviors from solutions and hydrogels were characterized. The nanocomponents (GO or RGO) were able to bind more drug molecules either for CPT or for doxorubicin hydrochloride (DXR) in solution. Therefore, GO or RGO composite hydrogel could potentially enable better controlled and gentler drug release (for both CPT and DXR) than native hydrogel.     

Characterization of nonderivatized plant cell walls using high-resolution solution-state NMR spectroscopy

A recently described plant cell wall dissolution system has been modified to use perdeuterated solvents to allow direct in-NMR-tube dissolution and high-resolution solution-state NMR of the whole cell wall without derivatization. Finely ground cell wall material dissolves in a solvent system containing dimethylsulfoxide-d(6) and 1-methylimidazole-d(6) in a ratio of 4:1 (v/v), keeping wood component structures mainly intact in their near-native state. Two-dimensional NMR experiments, using gradient-HSQC (heteronuclear single quantum coherence) 1-bond (13)C--(1)H correlation spectroscopy, on nonderivatized cell wall material from a representative gymnosperm pinus taeda (loblolly pine), an angiosperm Populus tremuloides (quaking aspen), and a herbaceous plant Hibiscus cannabinus (kenaf) demonstrate the efficacy of the system. We describe a method to synthesize 1-methylimidazole-d(6) with a high degree of perdeuteration, thus allowing cell wall dissolution and NMR characterization of nonderivatized plant cell wall structures.     

Synthesis of Ordered Biosilica Materials

Biogenic silica with amazing diversity of nanostructure shells,fibers and granules in diatoms and sponges is mediated by proteins and polysaccharides and forms at ambient pressure and temperatures.Chemical synthetic methods,in contrast,have to rely on extreme pH and /or surfactants to induce the condensation of silica precurors into specific patterns.One kind of benign synthesis method through plant cell wall template-directed ordered biosilica materials under ambient conditions in intriguing in this context.Organized silica materials in intercellular spaces of epidermal cells of tall fescue leaves were synthesized through molecular recognition between Si-OH and polysaccharide-OH or glycoprotein-OH of main components of plant cell walls and cellular processing as well when Si(OEt)4 was supplied rather than monosilicic acid.The biosynthesis of structural silica in tall fescue plant was correlated with the Si species applied,reflecting the slower condensation from tetraethoxysilane (TEOS) and thus providing greater opportunities for structural control by the underlying matrix of cell walls.The composition was estimated by energy dispersive X-ray(EDX) spectra on a scanning electron microscope.All organized structures showed carbon,oxygen and silicon peaks,indication that their formations differ from natural siliceous process.     

Exact determination of phagocytic activity of alveolar macrophages toward polymer microspheres by elimination of those attached to the macrophage membrane

A method for exact determination of phagocytic activity of alveolar macrophage (M) cells toward synthetic microspheres (MS) by optical microscopy was developed. We examined the effectiveness of the treatment of M samples with trypsin, acid or xylene to remove the polystyrene latex microspheres (PSL MS) attached to M cell membranes during their phagocytosis by M cells. We found that centrifugation, which was employed to collect M samples after incubation with MS, affected significantly the efficiency of the various treatments. Of the three treatments, xylene treatment without centrifugation was the most effective to determine the phagocytic activity of M cells, as xylene dissolved the PSL MS on the cell surface almost completely. This treatment was also effective in the case of poly(lactic-co-glycolic acid) MS (PLGA MS), which have been commonly used as an efficient vehicle for drug delivery system.     

Effect of thermal annealing on a bilayer polyvinyl alcohol/polyacrylic acid electrospun hydrogel nanofibres loaded with doxorubicin and clarithromycin for a synergism effect against osteosarcoma cells

Polyvinyl alcohol/polyacrylic acid (PVA/PAA) bilayer hydrogel nanofibres were successfully fabricated by electrospinning and physically crosslinked via heat treatment. The effects of the thermal annealing process on the structure, morphology, swelling, thermal properties and hydrophilicity of electrospun nanofibres were investigated. In addition, these membranes were also used to incorporate doxorubicin and clarithromycin for osteosarcoma treatment, one in each layer. These drugs were used because it is hypothesized in this work that a synergism occurs between both drugs. So, these membranes were analyzed towards their dual-drug release and potential cytotoxicity towards the U2OS human osteosarcoma cell line. Moreover, the water contact angle, disintegration, swelling and weight loss studies confirmed the rapid swelling and improved water stability of the annealed PVA/PAA bilayer nanofibres. The annealed bilayer nanofibres exhibited an increase in the average diameter and degree of crystallinity. In addition, the results revealed that a variation occurred in the degree of hydrophilicity of annealed PVA/PAA bilayer nanofibres. The PAA nanofibres surface exhibited higher hydrophilicity than the PVA nanofibres surface. Drug delivery presented to be as fast rate release for clarithromycin and slow-rate release for doxorubicin, which may be advantageous because both drugs exhibited to be synergetic for certain dosages presenting the combination of the drugs higher than 50% of cell inhibition, while these membranes had higher inhibition values (up to 90%), which was attributed to the PAA but also the drugs. These unique properties are of potential interest in drug delivery applications for dual drug delivery where the tunability of surfaces is desirable.     

Clerodane Diterpenoids from an Edible Plant Justicia insularis: Discovery,Cytotoxicity, and Apoptosis Induction in Human Ovarian Cancer Cells

Objectives: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics. Drug discovery from edible and medicinal plants represents a promising approach towards finding safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J. insularis and investigate their underlying mechanism of action. Methods: The cytotoxicity of the plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using column chromatography including HPLC, and the isolated natural products were characterized using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME and SwissTargetPrediction web tools were used to predict the molecular properties and possible protein targets of identified active compounds. Key finding: The two cytotoxic compounds were identified as clerodane diterpenoids: 16(α/β)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound 1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more potent than compound 2 with IC₅₀ values in the micromolar range against OVCAR-4 and OVCAR-8 cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound 1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2 are both drug like, and compound 1 may target various proteins including a kinase. Conclusions: Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via the induction of apoptosis, providing an abundant and valuable source of hit compounds for the treatment of ovarian cancer.     

Immobilization of plant cells in hybrid sol-gel materials

Cells of three different plant species were immobilized on a glass fiber fabric by sol-gel deposition. The process involved the following steps: (1) reinforcement of glass-fiber supports by coating with a gelling solution of hybrid-SiO₂ precursors, (2) entrapment of cells by stuffing the voids of the support with a suspension cell culture, (3) achievement of a definite immobilization by a primary treatment with SiO₂-sol, followed by gas phase reaction of tetraethoxysilane and diethoxymethylsilane with OH groups of cell wall and of surface silica. Immobilized cells maintained their viability as tested by the positive reaction to TTC and by the development of calli from stretched samples. The samples did not release cells in solution over a time period of four months, at least. The biosynthetic capability of one of immobilized species, Coronilla vaginalis, was studied by periodically monitoring the production of umbelliferone and marmesin which constituted the major secondary metabolites produced by in vitro cultured cells of this species. The results were evaluated in order to determine the versatility of the method and its potential for exploitation in continuous industrial-scale production of rare and fine chemicals.Abbreviations 2,4-D = 2,4-dichlorophenoxyacetic acid - K = kinetin - IAA = indol-3-acetic acid - NAA = naphthalenacetic acid - B5 = Gamborg's medium - MS = Murashige and Skoog medium - TEOS = tetraethoxysilane - DEMS = diethoxymethylsilane - DEDMS = diethoxydimethylsilane - TTC = tetrazolium salt     

Combination treatment for osteosarcoma with baculoviral vector mediated gene therapy (p53) and chemotherapy (adriamycin)

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been evaluated as a vector for gene delivery to human tumor cells. A human osteogenic sarcoma cell line, Saos-2, was found to be highly susceptible to infection with a baculoviral vector, with nearly 100% of Saos-2 cells being able to express a lacZ reporter gene after a brief exposure to the virus at a m.o.i. of 30 pfu/cell. The production of beta-galactosidase protein was 18-times greater than that in HepG2 cells which were previously thought to be the mammalian cells most susceptible to the baculovirus. The possibility of developing a baculovirus as a cytotoxic vector for p53-defective cancer was tested by destruction of Saos-2 cells (p53-/-) with a recombinant baculovirus containing the wild type p53 gene (BV-p53) in vitro. The p53 baculovirus induced apoptotic cell death in tumor cells in a dose-dependent manner with approximately 60% killing at an m.o.i. of 160 pfu/cell. Combined treatments of gene therapy (p53) and chemotherapy (adriamycin) resulted in synergistic and potent killing of the osteogenic sarcoma cells. For example, greater than 95% of Saos-2 cells were killed by the combination of BV-p53 (m.o.i. of 100) and adriamycin (35 ng/ml), whereas approximately 50% and approximately 55% cells were killed by BV-p53 and adriamycin alone, respectively. These results indicate that a baculoviral gene delivery vector can be used to efficiently target certain types of mammalian cells and the combination treatment of gene-therapy mediated by a baculovirus and chemotherapy may enhance induction of apoptosis in cancer cells.     

Non‐Newtonian Polymer–Nanoparticle Hydrogels Enhance Cell Viability during Injection

Drug delivery and cell transplantation require minimally invasive deployment strategies such as injection through clinically relevant high‐gauge needles. Supramolecular hydrogels comprising dodecyl‐modified hydroxypropylmethylcellulose and poly(ethylene glycol)‐block‐poly(lactic acid) have been previously demonstrated for the delivery of drugs and proteins. Here, it is demonstrated that the rheological properties of these hydrogels allow for facile injectability, an increase of cell viability after injection when compared to cell viabilities of cells injected in phosphate‐buffered saline, and homogeneous cell suspensions that do not settle. These hydrogels are injected at 1 mL min^?1 with pressures less than 400 kPa, despite the solid‐like properties of the gel when at rest. The cell viabilities immediately after injection are greater than 86% for adult human dermal fibroblasts, human umbilical vein cells, smooth muscle cells, and human mesenchymal stem cells. Cells are shown to remain suspended and proliferate in the hydrogel at the same rate as observed in cell media. The work expands on the versatility of these hydrogels and lays a foundation for the codelivery of drugs, proteins, and cells.     

Nano‐structured Hollow Carbon Materials from a Non‐isothermal Chemical Vapor Deposition of Polyphenols

Pyrogallic acid (PG) was used as a modeling carbon source in fabricating nano‐structured hollow carbon materials (HCMs) by a chemical vapor deposition (CVD) method. We found that non‐isothermal deposition can improve the integrity of the obtained HCMs. The different pyrolyzed species from PG under varied temperatures lead to the temperature‐dependent deposition yield, graphitization degree and morphology of the HCMs. HCMs including hollow spheres of varied sizes, cubic boxes with yolk‐shell structure, nanotubes, mesoporous particles and double‐shelled fibers, were prepared by using different templates, demonstrating the universality of this strategy. The carbon source has been extended to other plant polyphenols. The abundant and renewable solid precursors for CVD method endow this strategy excellent operation safety, improved storage and transportation convenience and low cost, and would boost the production of morphology‐ and size‐controlled HCMs and their applications in the fields such as water treatment, electrode materials, adsorbent, drug delivery, and so forth.     

Shear sensitivity of plant cells in suspensions present and future

Plant cells are a source of pharmaceuticals, fragrances, flavors, and dyes that are traditionally produced by extraction of tissues from whole plants. Recent trends in plant product research, transformed cell lines, and conservation policies place increased demand on plant cell culture technology. Unlike processing of microbial and animal cells in bioreactors, no economically viable process based on the suspension culture of plant cells in bioreactors has yet been possible in North America. It is proposed that the suspended-cell bioreactor is the method of choice and that plant cells respond to fluid forces (defined as laminar shear and turbulent eddies-based and bubble-based forces) differently from their animal cell counterparts in bioreactors. Although plant cells produce a tough cell wall, fluid forces, although not lethal within normal range, impact the membrane transport processes and metabolic function of plant cells; these effects are termed sublytic. Previous approaches to shear sensitivity of plant cells are reviewed in the context of these sublytic effects. A model for systematic evaluation of fluid-mechanical causes and physiological mechanisms behind sublytic effects is proposed. It is further proposed that, once understood, the plant cell’s sublytic responses to fluid force can be used advantageously in stirred suspension cultures.     

Activity and survival of spray-dried Beijerinckia sp. Microencapsulated in different carbohydrates

This study examined the possibility of preserving Beijerinckia cultures by encapsulation using a spray drier, for use in biotechnological processes in the production of biopolymers. An adequate choice of the wall (coating) material is one of the factors that will determine the degree of cell survival and the maintenance of fermentative activity in the encapsulated inoculum. Malt dextrin, dehydrated glucose syrups, modified starch, and acacia (gum arabic) were used as wall materials. The results showed that spray-dried Beijerinckia encapsulated in malt dextrin, stored for 2 mo, and inoculated into sterile must after rehydration presented the greates stability with respect to fermentative activity, although the glucose-encap sulated cells showed the highest percentage of viability during spray drying and during the storage period.