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by two models, namely h2 = c2t or h = c√t (1) INTRODUCTION Wickability of fabrics has become an important test as it discloses information on comfort, dyeability and usefulness as a sportswear. A number of papers on the wickability of yarns and fabrics have been published and reviews have appeared [1]. The role of water in transporting moisture has been appreciated for a very long time. A considerable amount of work has been done on the application of sericin to polyester and cotton fabrics with a view to conferring antimicrobial property to them [2][3][4]. From the papers published it is found that wickability test, although was performed on the fabrics, has not been studied in detail. Wicking is the spontaneous transport of a liquid driven into a porous system by a capillary force [5]. Wicking height is proportional to root of time. Lucas-Washburn equation, which is a very popular one, includes properties such as surface tension, radius of the capillary, contact angle and viscosity of the liquid which has been used to study wickability. It is reported that the weft density pore size and the arrangement of void spaces in fabric have a significant effect on the wicking performance [6]. It is also reported that the motion of liquid in the void spaces between fibers in a yarn impacts the mechanism of fabric wicking critically [7]. It is found that the rate of movement of liquid is governed by the fibre arrangement in yarn which control the capillary size and continuity [8]. Validity of Washburn's | 3,068,900 | 201239749 | 0 | 16 |
equation can be checked by two models, namely Where h is wicking height, t -time and k -time exponent, c and c' are constants. In this communication, the wickability of sericin treated polyester fabrics is dealt with. Although some data on wickability have been provided, they were not examined in detail. The applicability of Washburn's equation is discussed for a series of polyester fabrics that have been treated with sericin. Modification of polyester fabric Polyester fabric sample was scoured to remove any impurities and it was pretreated with alkali 1M (40 g/l) NaoH at 80°C for 45 min with 1:100 material to liquor ratio to create functional groups on its surface, before applying sericin to the fabrics. Application of sericin Sericin was applied on modified polyester fabric with and without the use of a crosslinking agent. 20 g/l of sericin solution was used. Alkali treated fabric were padded with the sericin solution in a laboratory padding mangle by a 2 dip 2 nip process. The padded fabric was dried at 80°C for 3 min and cured at 130°C for 2 min. Cured samples were then washed and dried. Glutaraldehyde was used as a crosslinking agent to attach sericin to alkali modified polyester. Plasma treated with DMDHEU The polyester fabric was prepared in the required dimension of 54×54 cm and weighed. This fabric was clamped to the frame and inserted in the plasma chamber between the two plates and pressure in the chamber was brought to 0 bar then the oxygen gas was passed to the chamber | 3,068,901 | 201239749 | 0 | 16 |
with the flow rate of 2 bar pressure. Initially the top side of the fabric was exposed to the plasma current 1.06 amp, plasma voltage 350 volt at temperature 29°C this was continued for 5 min. Then the bottom side of the fabric was exposed to the plasma current 1.53 amps, plasma voltage 300 volt at a temperature of 29°C for 5 min. After the process, the fabric was weighed again to determine the weight loss percentage. The plasma treated fabric was then wetted in water along with Turkey Red Oil 2 g/l and immersed in the prepared solution (sericin 25% (owf) and Dimethylol Dihydroxy Ethylene Urea 150% (owf), polyethylene emulsion 2g/l based on weight of the sample) for dipping process and was carried out using material-toliquor ratio of 1:9. This fabric was then padded in the 2dip-2nip padding mangles and curing process carried at the temperature of 140°C for 3 min. Plasma treatment changes the surface properties of the fabric [9]. Plasma treated with Glutaraldehyde (GA) The required dimension of the plasma treated fabric was weighed and the fabric was wetted in water along with wetting agent (TRO) and then treated with the solution of Sericin 25% (owf),GA 20 g/l,magnesium chloride 10 g/l and acetic acid 1.0 ml/l using material-to-liquor ratio of 1:9. The above procedure was followed for both padding and curing. Alkali treatment with DMDHEU and Glutaraldehyde The same untreated polyester fabric was treated with 15% NaOH (owf) with the material-to-liquor ratio kept at 1:40, at 60°C for 30 min. This alkaline treated | 3,068,902 | 201239749 | 0 | 16 |
polyester fabric was then treated with sericin, Glutaraldehyde, magnesium chloride and acetic acid and sericin, DMDHEU, polyethylene emulsion combination as in the same manner above and then padded and then cured. Untreated polyester with DMDHEU and Glutaraldehyde Untreated polyester fabric was directly treated with DMDHEU with other chemicals and Glutaraldehyde with the above mentioned chemicals. Drying and curing were carried out at 140°C for 3 min. Experimental In this study, seven samples of polyester fabric such as polyester fabric treated with alkali (PA), untreated polyester treated with sericin and Glutaraldehyde (USG), polyester fabric with sericin and DMDHEU (USD), Polyester fabric treated with alkali followed with Sericin and Glutaraldehyde (ASG), polyester fabric treated with alkaline followed with sericin and DMDHEU (ASD), Polyester fabric treated with plasma followed with sericin and Glutaraldehyde (PSG), Polyester fabric treated with plasma followed with sericin and DMDHEU (PSD). Details of the polyester fabrics used are given in Determination of Wickability of treated and untreated fabrics Wickability was studied by using vertical wicking method (DIN 53924 standard) as shown in figure 1. RESULTS AND DISCUSSION By plotting h 2 against t and using regression model passing through origin it will be possible to obtain values of K and check the applicability of Washburn's equation. The evaluation of the h 2 as a function of time is determined for given times in the region of 0-600 s and the slopes are given in table 2. The curve obtained is linear and the experimental values lead to a linear regression coefficient of R 2 exceeding | 3,068,903 | 201239749 | 0 | 16 |
0.99. It is necessary to get a correlation coefficient of more than 0.99, as only then will the Lucas Washburn's equation is followed. The results of the wicking test are shown in tables 2 and 3. Model A Plotting height in cm 2 against the time sec gives the following values which are given in table 2. Regression analysis has been done to get slope and intercept. Model B Values of slopes and intercepts are given in table 3, There are two models which are used to find out the validity of Washburn's equation. The first model is Where h is wicking height in cm and t -time in second. By plotting h 2 against t and using regression model passing through origin it will be possible to obtain values of k and check applicability of Washburn's equation. Values are shown in table 2. The second model B is h = c't k . This was proposed by Laughlin et al. [10] who suggested the following equation and Deboer [11] has also used this equation. It is interesting to note that Deboer [11] has not referred to Laughlin etals [10] paper in his study. By taking logarithm on both sides This model has been used by Nyoni [12] and Zhuang et al. [13] in their studies. Table 3 obtained by taking logarithm of wicking height values. Thus at 0 time, there is wicking which looks absurd. Another problem with regard to this model is that when wicking height and time have values less than 1, negative | 3,068,904 | 201239749 | 0 | 16 |
values are obtained. In this model when K = 0.5, it is taken that Washburn's equation is valid. Alternatively, the model h 2 = c 2 t is sound as for 0 time, 0 is the wicking [14][15][16]. This model is devoid of the deficiency as mentioned above. C O N C L U S I O N Using the model h 2 = c 2 t the experimental results have shown that the wicking height square had a positive and high correlation with time in the warp direction (R 2 = 0.99) indicating that the Lucas -Washburn's equation was suitable for evaluating the wicking property of sericin treated polyester fabrics. This other model namely, h = c't k is not sound as there are strange units. | 3,068,905 | 201239749 | 0 | 16 |
Low-Frequency Low-Intensity Ultrasounds Do Not Influence the Survival and Immune Functions of Cultured Keratinocytes and Dendritic Cells Low-frequency ultrasounds (US) are used to enhance drug transdermal transport. Although this phenomenon has been extensively analyzed, information on US effects on the single skin cell components is limited. Here, we investigated the possible effects of low-frequency US on viability and immune functions of cultured human keratinocytes and dendritic cells (DC), skin cells involved in the regulation of many immune-mediated dermatoses. We demonstrated that US, employed at low-frequency (42 KHz) and low-intensity (0.15 W/cm2) values known to enhance drug and water transdermal transport, did not affect extracellular-signal-regulated-kinase (ERK)1/2 activation, cell viability, or expression of adhesion molecules in cultured keratinocytes. Moreover, US at these work frequency and intensity did not influence the keratinocyte expression and release of immunomodulatory molecules. Similarly, cultured DC treated with low-frequency low-intensity US were viable, and did not show an altered membrane phenotype, cytokine profile, nor antigen presentation ability. However, intensity enhancement of low-frequency US to 5 W/cm2 determined an increase of the apoptotic rate of both keratinocytes and DC as well as keratinocyte CXCL8 release and ERK1/2 activation, and DC CD40 expression. Our study sustains the employment of low-frequency and low-intensity US for treatment of those immune skin disorders, where keratinocytes and DC have a pathogenetic role. Introduction Skin is a very efficient barrier against external agent penetration. Low permeability is attributed to the stratum corneum, the outermost skin layer, characterized by a robust structure containing dense dead cells, corneocytes, which are embedded in a | 3,068,906 | 14593905 | 0 | 16 |
continuous matrix of lipid bilayers. The tightly packed stacks of lipid lamellae in the extracellular space make the stratum corneum a highly impermeable membrane [1]. Low permeability of the skin is also a limit for the transdermal drug delivery, which could represent a valid alternative to oral delivery and injection. A diverse spectrum of mechanical [2,3], electrical [4], and chemical [5] techniques have been previously explored to enhance skin permeability and, consequently, to increase the transdermal drug transport. Exposure to ultrasound (US) has also been shown to greatly enhance the permeability of skin for permitting transdermal drug delivery, a phenomenon termed sonophoresis [6][7][8]. Based on the frequency used, three types of US can be applied to increase skin permeability: therapeutic US (from 1 to 3 MHz), high-frequency US (above 3 MHz), and lowfrequency US . However, low-frequency US have been found to be more efficient than high-frequency US in inducing the transdermal transport [7,8]. Previous studies have demonstrated that the generation of gas bubbles in the skin (cavitation) is the main mechanism responsible for sonophoresis and is better induced by low-frequency US, being inversely related to US frequency [9,10]. The efficacy of low-frequency US in inducing skin permeation is proportional to their intensity. US intensities starting from 0.1 W/cm 2 are sufficient to permit the transdermal transport across human skin of many permeants, including estradiol, salicylic acid, corticosterone, and water [7,[11][12][13]. Other than representing the major barrier between the body and the external environment, the skin provides a complex microenvironment for the initiation and shaping of | 3,068,907 | 14593905 | 0 | 16 |
T cell-mediated immune responses against microorganisms. Among skin cell components, keratinocytes and dendritic cells (DC), including epidermal Langerhans cells and dermal DC, are importantly involved in the induction and amplification of immune responses during inflammatory skin reactions [14][15][16]. In particular, DC are specialized to recognize and capture foreign antigens as well as to activate naïve T cells, and resident keratinocytes contribute to attract and retain discrete T-cell subsets into the skin. In order to test whether US application could influence the pathophysiological functions of skin cell components, we investigated on the effects of low-frequency US (42 KHz) administered at intensity ranging from 0.15 to 7 W/cm 2 , on cultured keratinocyte and DC populations. In particular, we examined the extracellular-signal-regulated kinase (ERK)1/2 activation, viability, and membrane molecule expression in US-treated keratinocytes, and, in parallel, their ability to produce inflammatory molecules, such as immune-modulatory membrane molecules, cytokines, and chemokines. Moreover, we explored the possible US effects on DC apoptosis, immune phenotype and functions, including activation of naïve T lymphocytes. US Generation and Application. The device generating low-frequency US has been realized by Teuco Guzzini technical laboratory (Montelupone, Macerata, Italy). US were produced continuously with a frequency of 42 KHz by means of piezoelectric ceramic vibrating at its own odd frequency and driven by a power supply. Piezoelectric ceramics were placed under an acrylic basement, used as a support for the cell culture plates, with an US transmission gel (Transound, EF Medica, Bolzano, Italy) film being interposed to ensure US distribution. The distance between the transducer ceramics and | 3,068,908 | 14593905 | 0 | 16 |
cell plate bottom was ∼ 0.3 cm. US emission was measured using a calibrated hydrophone placed inside the culture medium. Treatments with US were conducted for 7-21 minutes (1-3 cycles) at intensity ranging from 0.15 to 7.0 W/cm 2 . Keratinocyte Cultures and Treatments. Normal human keratinocytes were obtained from skin biopsies of healthy volunteers (n = 3), as previously reported [17]. Briefly, biopsies were disaggregated to single-cell suspensions by using six sequential treatments with 0.05% trypsin/0.02% EDTA (InVitrogen Life Technology, Carlsbad, CA, USA), each conducted for 30 minutes at 37 • C. Keratinocytes were then counted and seeded in F75 flasks on a fibroblast 3T3 feeder layer previously treated with mitomycin C (Sigma-Aldrich, Milan, Italy). Second or third passage keratinocytes were used in all experiments, with cells cultured in 3 cmdiameter plates in serum-free medium (Keratinocyte Growth Medium, Clonetics, Walkersville, MD, USA), for at least 3-5 days before performing experiments. Keratinocytes were treated with US at a frequency of 42 KHz for 1 cycle of 7 minutes or 3 cycles (in total 21 minutes). Some keratinocyte cultures were in parallel stimulated with 200 U/mL IFN-γ plus 50 ng/mL TNF-α (R&D Systems, Abingdon, Oxon, United Kingdom) for 24 hours. DC Preparations. DC were generated by culturing monocytes, previously obtained from the adherent fraction of peripheral blood mononuclear cells (PBMC) depleted of CD2 + , CD19 + , and CD56 + cells by monoclonal antibody (Ab)-coated immunomagnetic beads, in complete RPMI 1640 medium (Cambrex, East Rutherford, NJ), supplemented with 10% fetal bovine serum (Sigma-Aldrich) in the presence | 3,068,909 | 14593905 | 0 | 16 |
of 200 ng/mL GM-CSF (PeproTech, Rocky Hill, NJ) and 200 U/mL IL-4 (R&D Systems) at 37 • C. Medium was replaced after 3 days and cells were used at day 7 of culture. This procedure gave > 97% pure CD1a + , CD14 − DC preparations. To induce maturation, lipopolysaccharide (LPS) (Sigma-Aldrich) was added at 50 μg/mL for the last 24 hours of DC culture. Enzyme-Linked Immunosorbent Assay (ELISA). Cellfree supernatants from resting or stimulated keratinocyte cultures were tested for CXCL10 content using the Ab pair, the purified monoclonal Ab 4D5/A7/C5 for coating and the biotinylated 6D4/D6/G2 (BD PharMingen, San Diego, CA). CCL2 and CXCL8 were measured with OptEIA kits (BD PharMingen), as per the manufacturer's protocol. TNF-α and IL-6 were assayed with DuoSet ELISA kits (R&D Systems). DC supernatants collected from cultures 24 hours after the US and LPS treatments were assayed for IL-12 p70 subunit, TNF-α, IL-6, and IL-10 contents using the DuoSet ELISA kits (R&D Systems). The plates were analyzed in an ELISA reader (model 3550 UV, Bio-Rad, Hercules, CA). Keratinocyte and DC cultures were performed in triplicate for each condition. Results are given as mean ng or pg/10 6 cells ± SD. Mixed Lymphocytes Reaction (MLR) Proliferation Assay. In order to obtain responder population for the primary MLR assay, T naïve lymphocytes were purified from allogeneic PBMC nonadherent fraction by using CD45RA immunomagnetic microbeads (Miltenyi Biotec, Germany), according to the manufacturer' instructions. At day 5 of culture, immature DC were left untreated or were treated with US at a frequency of 42 | 3,068,910 | 14593905 | 0 | 16 |
KHz for 1-3 cycles before adding LPS for the next 24 hours. Untreated or US-treated mature DC were then washed and cocultured in 96-well flat bottomed microplates in serial dilutions (0.625 to 10 × 10 3 /well) together with purified allogeneic T naïve lymphocytes (2 × 10 5 /well) in complete RPMI medium. Cocultures were pulsed at day 5 with 2 μCi/mL [ 3 H]-thymidine (Amersham, Little Chalfont, UK) for about 16 hours and then harvested onto fiber-coated 96-well plates. Radioactivity (counts per minute, CPM) was measured in a β-counter (Topcount; Packard, Groningen, The Netherlands) and results were given as the mean counts per minute ± SD of triplicate cultures. Western Blotting and Densitometry. Protein extracts were prepared by solubilizing cells in RIPA buffer (1% NP-40, 0.5% sodium dehoxycholate, 0.1% sodium dodecyl sulfate, SDS) containing a mixture of protease and phosphatase inhibitors. Proteins were subjected to SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The latter were blocked, and probed with anti-ERK1/2 (C16; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-ERK1/2 (E4; Santa Cruz Biotechnology), or with anti-βactin (C11; Santa Cruz Biotechnology) Abs diluted in PBS containing 5% nonfat dried milk. Filters were developed using the ECL-plus detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK), and, then, subjected to densitometry using an Imaging Densitometer model GS-710 (Bio-Rad) supported by the Molecular Analyst software, and band intensities were evaluated in three independent experiments. The densitometry values of phopsho-ERK1/2 were divided by the values of total ERK1/2 and, then, to βactin bands, and expressed as -fold induction (F. I.) ± | 3,068,911 | 14593905 | 0 | 16 |
SD in experimental relative to untreated samples, to which were given a value of 1. Statistical Analysis. The Wilcoxon's signed rank test was used (SigmaStat, Jandel Co., San Rafael, CA) to compare the apoptosis rate as well as the expression levels of membrane molecules, phospho-ERK1/2, chemokines, and cytokines between unstimulated and US-treated cells. P values ≤ 0.05 were considered significant. Although several studies have examined the effects of US application on whole skin [9,11], evidence of US-induced responses on the single cell components of the skin is still limited. To this end, the effects of low-frequency US were analyzed on keratinocyte cultures established from skin biopsies of healthy donors. US were employed on cell cultures at frequency (42 KHz) and intensity (0.15 W/cm 2 ) values known to be effective in enhancing human skin permeation in vivo [12]. We firstly investigated on the US effects on keratinocyte expression and activation of ERK1/2 proteins, which sustain proliferative and self-protective programs in keratinocytes [18]. As shown in Figure 1, the phosphorylation status of ERK1/2 did not vary between untreated and US-treated keratinocytes, although it was upregulated upon IFN-γ and TNF-α treatment, as previously described [18]. Consistently, the levels of ERK1/2 total proteins were unaffected by US irradiation. We, then, analyzed keratinocyte viability by evaluating annexin V exposure on plasmamembrane and propidium iodide (PI) incorporation into DNA or caspase 9 expression. As positive control of apoptosis, keratinocyte cultures were in parallel stimulated with IFN-γ plus TNF-α. As shown in Figure 2(a), upon 1cycle or 3-cycle US stimulation the number | 3,068,912 | 14593905 | 0 | 16 |
of apoptotic cells (lower and upper right quadrants) or necrotic cells (upper left quadrant) was comparable to that observed in untreated cultures. In contrast, an high number of apoptotic and necrotic cells were induced by the IFN-γ plus TNF-α treatment. The keratinocyte apoptotic rate was also assessed by measuring the level of active caspase 9, a proteolytic enzyme involved in the early phase of apoptotic processes, whose expression was revealed using the fluorescein-labeled inhibitor FAM-LEHD-FMK. Stained apoptotic cells were analyzed by flow cytometry and, as shown in Figure 2(b), only IFN-γ plus TNF-α treatments were able to induce an high percentage (85%) of caspase 9-positive cells. In contrast, both 1-cycle and 3-cycle US did not modify viability of cells (Figure 2(b)). Moreover, treatment of keratinocytes with 1 or 3 cycles of US at 42 KHz did not alter the surface expression of the β1 and α6β4 integrins, essential components of hemidesmosomes involved in the cell-cell and cell-extracellular matrix adhesion of keratinocytes in the epidermis [19] (Figure 3). Similarly, ICAM-1 and HLA-DR, two membrane molecules involved in the retention and activation of T lymphocytes during skin inflammatory responses were also unaffected by US treatment, whereas they were highly upregulated by IFN-γ and TNF-α, as previously reported [14]. Chemokine and Cytokine Release by Human Keratinocytes Is Not Modified by US at 42 KHz and 0.15 W/cm 2 . Keratinocytes can be activated by a variety of stimuli to produce chemokines and cytokines important for the recruitment and activation of immune cells and for the formation of a T | 3,068,913 | 14593905 | 0 | 16 |
cell-rich infiltrate during skin diseases [14,15]. In the next series of experiments we tested whether US stimulation could influence the release of chemokines, such as CXCL10, CCL2, and CXCL8, abundantly expressed by keratinocytes activated by IFN-γ and mostly by IFN-γ plus TNF-α. US-treated keratinocytes cultures were also analyzed for the release of the inflammatory cytokines IL-6 and TNF-α. Figure 4 shows that both 1-cycle and 3-cycle US at a frequency of 42 KHz did not influence the release of the proinflammatory chemokines CXCL10, CCL2 CXCL8 (Figures 4(a)-4(c)) and the cytokines IL-6 and TNF-α (Figures 4(d) and 4(e)), all abundantly upregulated by keratinocytes when activated with IFN-γ or IFN-γ plus TNF-α. Treatment with US at 42 KHz and 0.15 W/cm 2 Does Not Induce Apoptosis and Necrosis of DC. The effects of the low-frequency US were also examined on DC, the skin cell component representing the main mediator of inflammatory responses during skin diseases [20]. DC can be generated in vitro from peripheral blood or bone marrow progenitors. In particular, circulating CD14 + monocytes, cultured in the presence of GM-CSF and IL-4, can acquire many phenotypical features of primary DC, such as an increased expression of immune membrane molecules, and become able to evoke potent T cell responses [16]. To investigate whether US application could influence the viability of DC population, we analyzed monocyte-derived DC irradiated with US at 42 KHz frequency and 0.15 W/cm 2 intensity by performing the trypan blue exclusion test or using the annexin V apoptosis Journal of Biomedicine and Biotechnology detection system. | 3,068,914 | 14593905 | 0 | 16 |
Trypan blue assay demonstrated that both 1 cycle and 3 cycles of US stimulation did not significantly vary the number of viable cultured DC (viability superior to 93%; data not shown). Similar results were obtained by performing annexin V and PI stainings. As shown in Figure 5, after 3-cycle US stimulation, the number of annexin V + or annexin/PI + apoptotic cells was similar to that observed for untreated cells (6.1% and 5.4% versus 7.9% and 5.3%, resp.). Similarly, no significant difference between US-treated and untreated cells was detected in the number of necrotic cells (5.4% versus 3.4%, resp.). These data exclude any deleterious effect of US treatment on DC viability and apoptosis. or US-treated mature DC were cocultured with purified naive T cells, and proliferation evaluated in terms of [ 3 H]thymidine incorporation. As shown in Figure 8, 1-3 cycle US stimulation did not influence the alloantigen-presentation function of mature DC, which induced proliferation rate in T cells similar to that observed for untreated cells. As a whole, these data indicate that DC phenotype and function are not altered by US stimulation. Enhancement of Intensity of Low-Frequency US Alters the Survival and Immune Responses of Keratinocytes and DC. Previous studies demonstrated that the transdermal transport rate across skin is proportional to US intensity, although high-intensity US can produce significant thermal effects leading to erythema and dermal necrosis [9]. In order to identify a threshold value of US intensity at which cell viability and immunological functions of cultured keratinocytes and DC were altered and/or compromised, we | 3,068,915 | 14593905 | 0 | 16 |
treated cells with low-frequency US at intensity ranging from 0.15 to 7 W/cm 2 . US-treated keratinocytes were, then, analyzed for the exposure of apoptotic markers, ERK1/2 activation and the expression of immune mediators. DC treated with US at different intensities were also examined for apoptosis and immune phenotype. As shown in Table 1, US applied at intensities up to 2.3 W/cm 2 did not substantially vary the apoptotic rate nor the levels of ERK1/2 activation in cultured keratinocytes. In contrast, at the intensity value of 5.0 W/cm 2 , the number of apoptotic cells started to increase (1.93-fold increase of apoptotic cell percentage versus untreated samples, Table 1), and at 7.0 W/cm 2 cell viability was greatly compromised (3.54-fold increase of apoptotic cell percentage versus untreated samples, Table 1). In parallel, we could observe a slight, but significant, increase of the adhesion molecules ICAM-1 and α6β4 integrin, but not of HLA-DR in keratinocytes treated with US at the highest intensity values (Table 1). Of note, keratinocytes treated with US at 5 and 7 W/cm 2 showed an increase of CXCL8 release as well as phospho-ERK1/2 expression (Table 1). In addition, no substantial differences of CXCL10, CCL2, IL-6, and TNF-α production could be observed in UStreated compared to unstimulated cells (Table 1). Similarly to keratinocytes, DC showed an enhanced cell mortality when treated with US at the highest intensities. In particular, apoptotic DC started to accumulate upon treatment with US at 5 W/cm 2 (1.94-fold increase compared to untreated DC, Table 1), and substantially increase at | 3,068,916 | 14593905 | 0 | 16 |
7.0 W/cm 2 (3.21-fold increase compared to untreated DC, Table 1). Irradiation with US at intensity of 5.0 and 7.0 W/cm 2 also determined a slight but significant increase of CD40 co-stimulatory molecule (Table 1). In contrast, we could not detect any variations in the expression of membrane CD1a, CD83, CD80, HLA-DR, and CD86 by US-irradiated compared to untreated DC (Table 1). Finally, the enhancement of intensity values of low-frequency US did not influence the release of the cytokines TNF-α, IL-6, and IL-12 by both immature (Table 1) and mature DC (data not shown). Discussion The skin represents an advantageous portal for drug delivery, and its exposure to US is considered as an useful tool to enhance the transdermal transport by increasing cutaneous permeability to a variety of therapeutics [6][7][8]. Although sonophoresis has been a topic of extensive research in the last 15 years, information on US biological effects on the single skin cell components is limited. In this study, we investigated whether application of low-frequency US influenced the physiological and immune functions of human keratinocytes and DC in vitro. Low-frequency US were used at frequency (42 KHz) and intensity (0.15 W/cm 2 ) values known to be effective in enhancing human skin permeation in vivo [12]. It has been widely documented that, during inflammatory skin reactions, following exposure to lymphokines, especially IFN-γ and TNF-α, epidermal keratinocytes become an important source of inflammatory molecules [14,15]. For instance, activated keratinocytes can express very high levels of membrane molecules, including MHC Class II and ICAM-1, as well as | 3,068,917 | 14593905 | 0 | 16 |
cytokines and chemokines, such as CXCL8, responsible for the intraepidermal collection of neutrophils, and CXCL10 and CCL2, both aimed at Tcell recruitment [14]. In association to resident skin cells, migrating immune cells play a central role, especially in the initiation of cutaneous inflammatory responses [16]. Among them, dermal DC, together with resident skin Langerhans cells, can generate strong primary immune responses. DC reside in an immature form inside unperturbed skin, where they are adapted for capturing pathogens and processing antigens, eventually becoming able to stimulate naïve T cell proliferation and to promote T helper or cytotoxic responses [16,20]. In this study, we demonstrated that application of US at 42 KHz and 0.15 W/cm 2 on cultured human keratinocytes did not perturb their normal viability nor induce the activation of ERK1/2 proteins, which are known to sustain proliferative and self-protective programs in keratinocytes [18]. In fact, the apoptotic and necrotic cell percentage as well as the ERK1/2 phosphorylation status did not vary between untreated and US-irradiated samples. In addition, we demonstrated that US at 42 KHz and 0.15 W/cm 2 did not alter their phenotype, evaluated in terms of surface expression of the β1 and α6β4 integrins, two proteins importantly involved in the formation of the epidermal structure [20], and of ICAM-1 and HLA-DR, two typical markers of immune-activated keratinocytes [14]. Moreover, US administered at 42 KHz and 0.15 W/cm 2 did not influence the release of the chemokines CXCL10, CCL2, and CXCL8, abundantly secreted by IFN-γ plus TNF-α-activated keratinocytes, as well as the release of the | 3,068,918 | 14593905 | 0 | 16 |
cytokines IL-6 and TNF-α. However, we could also demonstrate that irradiation of keratinocytes with low-frequency US set at intensity values higher that 0.15 W/cm 2 , namely, at 5 and 7 W/cm 2 , determined substantial changes in survival and immune molecule expression in these cells. In fact, in these conditions, keratinocytes increased the apoptotic process and also the expression of the adhesion molecules ICAM-1 and α6β4 integrin. Of note, at the US intensity value of 5 and 7 W/cm 2 , CXCL8 and phospho-ERK1/2 were also upregulated in keratinocytes. Since both CXCL8 and phospho-ERK1/2 sustain keratinocyte growth and proliferation [14,15,18], the observed increase of these proteins could be evoked to counteract the proapoptotic/necrotic effects induced by US at high intensities. All these data support and are consistent with previous findings showing the safety of the low-frequency US at low intensity values. In fact, Boucaud et al. using optical and electron microscopy, showed that human skin samples exposed to US at intensities lower [21]. In addition, no substantial structural changes and modifications were found in skin treated with US to enhance the topical delivery of tetanus toxoid [22] and antisense oligonucleotides [23]. Recently, it has been demonstrated that there is no difference in morphology and in mitogenic activities between US-treated and untreated skin-derived fibroblasts [24]. On the other hand, the enhancement of intensity of lowfrequency US did not alter either the expression of HLA-DR, CXCL10, CCL2 or the proinflammatory cytokines IL-6 and TNF-α. Indeed, previous findings demonstrated that US at a frequency of 1 MHz could | 3,068,919 | 14593905 | 0 | 16 |
upregulate both IL-1α and TNF-α in mouse epidermal keratinocytes in vivo, through a mechanism involving the enhancement of local calcium release [25]. This cytokine upregulation can also be due to the fact that mice skin is extremely sensitive to US, even at a low frequency, and responds to US with important structural damages, including the detachment of the outer layer of the stratum corneum and pore formation [26], in their whole responsible for the epidermal upregulation of inflammatory cytokines [27]. Similar to human keratinocytes, DC showed unaffected viability, immune function, and phenotype in response to US at 42 KHz and 0.15 W/cm 2 . In fact, annexin V/PI analysis allowed us to exclude any cytotoxic or proapoptotic effect of US on DC. In parallel, we found that low-frequency lowintensity US treatment did not influence the DC expression of the membrane molecules CD40, CD80, CD83, CD86, HLA-DR, and CD1a, or release of cytokines involved in DC maturation. The latter include IL-6, TNF-α, IL-12, and IL-10, which are crucial for driving differentiation of naïve T cells toward the IFN-γ-producing type 1 phenotype [20]. DC maturation correlates with their ability to present antigens to T lymphocytes, and, indeed, we demonstrated that the treatment with US at 42 KHz and 0.15 W/cm 2 did not impair the DC immunostimulatory activity, as assessed by analysing the proliferation of allogeneic T lymphocytes in MLR assay. To our knowledge, these findings are the first demonstration that low-frequency US do not potentially alter the immune responses induced by DC and sustain the employment of | 3,068,920 | 14593905 | 0 | 16 |
US as neutral transducer for the transcutaneous immunization [21]. In fact, US have been used as physical adjuvant to enhance the delivery of vaccines (i.e., tetanus toxoid) into the mouse skin and, thus, to generate potent systemic immune responses [21]. In this vaccination system, other than facilitating immunogen penetration, US appeared to activate Langerhans cells, the antigen professional DC residing in the epidermis. Indeed, Langerhans cells could be induced to undergo maturation by the apoptotic bodies locally released by dying cells rather than a direct effect of US. Consistently, another study reported that barrier disruption induced Langerhans cell activation without migration to regional lymph nodes [28]. By contrast, the enhancement of US intensity to 5 and 7 W/cm 2 caused a substantial increase of DC apoptotic rate. In these conditions, DC also showed a slight but significant increase of the expression of the co-stimulatory molecule CD40, and no changes in the levels of membrane CD1a, CD80, CD83, CD86, HLA-DR, nor of secreted IL-12, IL-6, and TNF-α. The upregulation of CD40 on DC membrane, not accompanied by the increase of the other stimulatory and co-stimulatory molecules, might not significantly influence T cell activation. However, further studies are needed to definitively establish whether lowfrequency US at high intensities can influence DC-mediated immune responses. In conclusion, the data collected in this paper demonstrate that US at low-frequency (42 KHz) and low-intensity (0.15 W/cm 2 ) do not influence cell viability and immune functions of both keratinocytes and DC in vitro. Therefore, our results sustain the safety of the in | 3,068,921 | 14593905 | 0 | 16 |
vivo application of US having these physical parameters, as well as their employment for the transdermal delivery of drugs active in those immune-mediated skin disorders where keratinocytes and DC have a pathogenetic role. However, the enhancement of the intensity of low-frequency US determines an increase of the apoptotic rate of both keratinocytes and DC as well as of keratinocyte self-protective responses and DC CD40 expression. We identify at 5 W/cm 2 the intensity value of lowfrequency US at which the viability and immune functions of both keratinocytes and DC are altered in vitro. In order to guarantee the safety of US application in vivo, further studies, performed by irradiating whole human skin with lowfrequency US at increasing intensities, and aimed at analyzing keratinocyte and DC expression of cell viability markers and immune-modulatory molecules, should be conducted. | 3,068,922 | 14593905 | 0 | 16 |
Effect of local bone marrow stromal cell administration on ligature-induced periodontitis in mice. Bone marrow-derived multipotent stromal cells (BMSCs) have potent antiinflammatory effects. This study aimed to investigate the antiinflammatory potential of BMSCs using a mouse model of ligature-induced periodontitis. BMSCs were isolated from the femurs and tibiae of mice. Periodontitis was induced by placing a ligature around the right maxillary second molar. After 3 days, the mice were administered BMSC in the gingiva of the mesial interdental papilla around the ligatured molar. The ligatured and non-ligatured mice that were not administered BMSC served as controls. Differences in inflammatory infiltration and bone resorption around the roots of the second molar were assessed and were subsequently quantified using microcomputed tomography (micro-CT), histological analysis, and tartrate-resistant acid phosphatase (TRAP) staining. Micro-CT revealed that alveolar bone loss around the ligatured molars increased in a time-dependent manner; however, the effect was significantly less in BMSC-treated mice compared with ligatured control mice. Tissue histopathology revealed that BMSC administration mitigated inflammatory infiltration in ligatured BMSC mice. In addition, the number of TRAP-positive osteoclasts was markedly elevated in ligatured control mice compared with those in BMSC-treated mice. These findings indicate that local BMSC administration can mitigate inflammation and alveolar bone resorption, suggesting that administering BMSC leads to new therapeutics for periodontitis. Introduction Periodontitis is a common chronic dental disease worldwide, and based on a national survey, it is present in 42.5% of Japanese citizens (1,2). The onset of this disease results from bacterial plaque accumulation that elicits an inflammatory cascade, leading to leukocyte | 3,068,923 | 5662686 | 0 | 16 |
infiltration, connective tissue destruction, pocket formation, and alveolar bone resorption (3)(4)(5). Although the initial inflammatory reaction is generally reversible as represented by gingivitis, an untreated disease can progress to a chronic state that is characterized by inflammation that extends from the gingival tissue to the adjacent alveolar bone and periodontal ligament. Because these periodontal structures play a critical role in supporting teeth, failure to treat periodontitis can result in tooth loss. The primary goal of currently applied therapeutics for periodontitis is reducing the subgingival bacterial load via instrumental debridement with surgical or non-surgical approaches (6,7). However, entire removal of the resulting biofilm when bacterially affected root surfaces are embedded in soft tissues or located in anatomically inaccessible areas is almost impossible; antibiotics or antiseptics are often administered as adjuvant treatments (8). Therefore, some periodontitis cases can be unresponsive to a conventional therapy, calling for the development of novel therapeutic regimens for periodontitis. Multipotent mesenchymal stromal cells (MSCs) are postnatal stem cells that are found in various human tissues. They possess self-renewing ability and can differentiate into several cell lineages, including osteocytes, chondrocytes, and adipocytes (9)(10)(11)(12). Most studies on MSCs have focused on their regenerative properties; however, recent studies have demonstrated the profound antiinflammatory effect of MSCs by secreting factors in response to activation cues from injured tissues (13,14). In addition, these cells can migrate to sites of injury and promote cell survival and proliferation via direct cell-tocell contact or paracrine activity. These actions, combined with their accessibility and in vitro expansion potential, make MSCs an attractive | 3,068,924 | 5662686 | 0 | 16 |
candidate for antiinflammatory treatments. As such, this study investigated the possible therapeutic effects of Bone marrow-derived multipotent stromal cells (BMSCs) in an experimental animal model of progression stage of periodontitis. Animals All animal experiments were performed according to the guidelines of The Animal Research and Care Committee at the Nihon University School of Dentistry. The Animal Experimentation Committee of Nihon University School of Dentistry approved the study protocols (AP15D027). Eight-week-old male wild-type C57BL/6 mice (CLEA Japan, Inc., Tokyo, Japan) were housed in an experimental animal room (22°C, 55% humidity, 12-h light/ dark cycle). The mice were divided into the following three groups (n = 45 total; 15 per group): 1) ligature + BMSCs, 2) ligature control (ligature + PBS vehicle administration), and 3) no ligation control. Isolation and culture of BMSCs BMSCs were isolated from the femurs and tibiae of 8-week-old C57BL/6 mice, as previously described (15). In brief, the femurs and tibiae were washed thrice in fresh PBS with vigorous shaking and then crushed with a sterile pestle and mortar, and the fragments were flushed thrice with 10 mL Hanks balanced salt solution. The flushed bone fragments were then transferred to a 50-mL conical tube and incubated with 20 mL preheated Dulbecco's modified Eagle's medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 0.2% (wt/vol) collagenase (Wako Pure Chemical Industries, Osaka, Japan) for 1 h. The resulting cell suspension was filtered through a 70-μm cell strainer and then placed on ice for 70 min to quench collagenase activity. After centrifugation, the pellet was treated | 3,068,925 | 5662686 | 0 | 16 |
with ice-cold sterile H 2 O for 6 s to lyse red blood cells, leaving only BMSCs in the resulting pellet. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C in a humidified atmosphere that contained 5% CO 2 , with daily medium changes. Cell surface marker analysis The phenotype of BMSCs was confirmed using flow cytometry for surface marker stem cell antigen-1 (Sca-1). Cells were harvested with trypsin and incubated with the Sca-1-FITC antibody (BD Biosciences, San Jose, CA, USA) for 30 min (16); they were then stained with 1 μg/ mL propidium iodide (Sigma Aldrich). Flow cytometry data were analyzed using the FlowJo software (Treestar, San Carlos, CA, USA). Induction of progression stage of periodontitis Eight-week-old male mice were intraperitoneally anesthetized using a mixture of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol tartrate (2.5 mg/ kg). To induce periodontitis, a 5-0 silk ligature was tied around the right maxillary second molar and sutured to prevent periodontal tissue damage (17). Ligatures were checked daily and maintained in place for all mice throughout the 14-day experimental period (n = 30). Body weights were recorded daily throughout the experimental period; no differences between the ligatured and non-ligatured control groups were observed. Determination of cell dosage and administration in mice We determined the optimal cell dose to prevent bone loss in this mouse model. In brief, three BMSC concentrations (10 4 , 10 5 , or 10 6 cells in 5 µL PBS) were administered into the gingiva of the | 3,068,926 | 5662686 | 0 | 16 |
mesial interdental papilla at the second molar using a 33-G syringe (Hamilton, Reno, NV, USA) at 3 days after ligature placement (n = 30 total; 15 per group). The bone loss was then monitored using microcomputed tomography (micro-CT) and histological analysis as described below. Micro-CT imaging and analysis The RmCT system (Rigaku Co., Tokyo, Japan) has a microfocus X-ray tube with a 7-µm focal point and a sensor with a 4-inch image intensifier. The X-ray source and image intensifier were connected by a basal plate, and the I-arm rotated on the vertical plane and was driven by a direct-drive motor. The mice were placed on the stage to image the regions of interest with exposure parameters at 90 kV and 100 μA (18)(19)(20). The size of the regions of interest was 1 × 1 × 3 mm. The bone volume (BV) in the regions of interest was measured to assess bone resorption. Images were reconstructed using the i-View software. Cortical BV in the regions of interest was measured using the micro-CT imaging data in the BV measurement software (Kitasenju Radist Dental Clinic I-View Image Center., Tokyo, Japan). For this, gray values and the corresponding number of voxels were calculated and compared with that from the first day of imaging at days 3, 5, 7, and 14 post-ligature placement. Tissue histology Inflammation and alveolar bone loss were assessed by hematoxylin and eosin staining. In brief, the tissue was harvested from euthanized animals and fixed in 10% neutral buffered paraformaldehyde for 48 h, decalcified in 15% EDTA for | 3,068,927 | 5662686 | 0 | 16 |
5 weeks, dehydrated using a graded ethanol series, and then embedded in paraffin. Specimens (n = 45 total) were sagittally sectioned (4-μm thick) with a microtome (Leica RM2165, Nussloch, Germany) and then stained with hematoxylin and eosin. For the quantitative analysis, five stained sections from each specimen were selected, and alveolar bone loss was quantified by measuring the distance between the cementoenamel junction (CEJ) and alveolar bone crest (ABC) in the mesial and distal roots of the maxillary second molar and between the roof and ABC in the furcation area using light microscopy (Eclipse LV100POL and Digital Sight DS-L2; Nikon, Tokyo, Japan). Gingival recession was also determined by measuring the distance between CEJ and the apical termination of the junctional epithelium. Tartrate-resistant acid phosphatase staining Osteoclasts and odontoclasts were defined as multinucleated (≥3) tartrate-resistant acid phosphatase (TRAP)-positive cells in contact with the surface of the alveolar bone and root cementum of furcation sections, respectively. At 4 and 11 days after BMSC administration (7 and 14 days post-ligature placement), osteoclasts and odontoclasts were detected by TRAP staining performed using a leukocyte acid phosphatase kit (Sigma Adrich) (21). Images were captured using a microscope (Eclipse LV100POL; Nikon, Tokyo, Japan), and TRAP-positive cells were counted using the Image J software (NIH, Bethesda, MD, USA). Localization of DiI-labeled BMSCs BMSCs (wild-type C57BL/6) were harvested with 0.25% trypsin-EDTA, resuspended at 10 6 cells/mL in DMEM, and then labeled with the fluorescent tracer dye DiI (5 μL/mL, Molecular Probes, Eugene, OR, USA) for 20 min. Stained cells were centrifuged at 280 g | 3,068,928 | 5662686 | 0 | 16 |
for 5 min and washed twice with PBS before administration into recipient mice. The survival and localization of BMSCs were then examined at 0 and 4 days after cell administration (19). Statistical analysis Statistical comparisons between the groups were performed using one-way analysis of variance, followed by Tukey-Kramer post hoc test. Statistical analysis was conducted using GraphPad Prism software version 6 (San Diego, CA, USA). Statistical significance was defined as P < 0.05. In vitro characterization of multipotent BMSCs BMSCs isolated from C57/BL6 mice were capable of expanding in vitro and formed fibroblast-like colonies after culturing for 7 days in an MSC growth medium (Fig. 1A). Flow cytometry showed that 40% of cells expressed the mesenchymal stem cell marker Sca-1 (Fig. 1B), consistent with a previous study (22). Therefore, the cultured cell populations were positive for key stem cell markers such as Sca-1. Ligature-induced periodontitis and BMSC efficacy Preliminary analyses were performed to determine the optimal cell number of BMSC administration (10 4 , 10 5 , or 10 6 ), including Sca-1-positive and Sca-1-negative cells for therapeutic efficacy. Bone loss was estimated using micro-CT and histological images in each cell number group at 7 days after ligature placement ( Fig. 2A). Quantification analysis demonstrated that the mice administered 10 6 BMSCs retained significantly more BV compared with those administered 10 4 or 10 5 BMSCs (Fig. 2B). Furthermore, based on the histological evaluation at day 7 after administration, a remarkable infiltration of mononuclear cells was observed in the furcation area of mice administered 10 4 | 3,068,929 | 5662686 | 0 | 16 |
or 10 5 cells, but not 10 6 cells (Fig. 2C). Because a significant favorable effect was observed in mice administered 10 6 BMSCs, we selected this dosage for our subsequent analyses. Periodontitis-induced bone resorption Micro-CT analysis showed no alveolar bone resorption in the no ligation control group at 14 days after placement, as expected (Fig. 3A). However, 50% of the alveolar bone in the mesial and distal roots was resorbed in the ligature control group at 5 days after ligature placement, whereas only 33% was resorbed in the ligature + BMSC group (Fig. 3B). Horizontal section analysis revealed that the buccal and distal root alveolar bones were completely resorbed at day 7 and 14, respectively, in the ligature control group. In comparison, the mesial root was still embedded within the alveolar bone in the ligature + BMSC group at day 14 (Fig. 3A). Moreover, BMSC-treated mice had markedly less bone loss than the ligature control mice after 5 days ( Fig. 3B; P < 0.01). Histological analysis Representative low-magnification histological images revealed a gradual decrease in alveolar bone height and complete resorption on day 14 in the ligature control group (Fig. 4A, B). Analysis of inflammatory cell infil- trate also showed a marked decrease in mononuclear cells in the medial and distal lamina propria under the gingival epithelium in the ligature + BMSC group compared with that in the ligature control group on day 5 (Fig. 4C). Moreover, principal gingival and periodontal ligament fibers were clearly observed in the mesial area of BMSC-treated mice but | 3,068,930 | 5662686 | 0 | 16 |
not in the ligature control mice (Fig. 4C). In addition, BMSC-untreated ligatured mice exhibited a marked increase in polymorphonuclear cells in the alveolar bone and root cementum surface within the furcation area, which were subsequently identified as osteoclasts and odontoclasts by TRAP staining (Fig. 5). Quantification of alveolar bone loss BMSCs from untreated, ligatured teeth showed significant increases in the CEJ-ABC and furcation-ABC distances throughout the experimental period consistently in our histological analysis. The ligature control group showed markedly greater CEJ-ABC distances than the ligature + BMSC group at 7 and 14 days after ligature placement (Table 1 and Fig. 6; double-headed arrows; P < 0.05). In addition, BMSC treatment significantly reduced gingival recession as determined by the distance between CEJ-the apical termination of the junctional epithelium (Table 2; Fig. 6B; double-headed arrows; P < 0.05). Data are expressed as mean ± SD (µm). n = 10 per group, *P < 0.05, **P < 0.01 by one-way ANOVA test. DiI-labeled BMSC tracking To determine the localization of BMSCs after administration, BMSCs labeled with DiI were administered in the gingiva of the mesial interdental papilla at the mesial root in the second molar of recipient mice. At 1 h and 4 days after BMSC administration, the tissue sections were harvested and examined for DiI emission. Notably, BMSCs were observed within the gingival epithelium at 1 h after administration, whereas BMSCs also appeared in the lamina propria of gingiva after 4 days (Fig. 7). Discussion This study aimed to investigate the effect of locally administered BMSCs, including Sca-1-positive | 3,068,931 | 5662686 | 0 | 16 |
cells, on periodontal tissue destruction in a mouse model of progression stage of periodontitis. Various animal models have been developed to understand the etiology of periodontal diseases and characterize potential therapies (23,24). Ligature-induced periodontitis is one of the most widely used evidence-based disease models (25). Our model clearly showed increased alveolar bone resorption and inflammatory cell infiltration, indicating that the findings were consistent with those from a previously reported mouse model of ligature-induced periodontitis (26). BMSCs can be administered by several methods such as systemic intravenous injection or local injection into the tissue of interest. Of these, intravenous injection is the most widely used because of the following advantages: 1) intravenous injection is minimally invasive, 2) BMSC infusions are reproducible, and 3) administered cells remain close to the oxygen-and nutrient-rich vasculature of the target tissue (27). However, many infused BMSCs may be trapped by some organs after intravenous injection, interrupting the homing of BMSCs to the target tissues. Direct BMSC administration is not the preferred for kidney and liver diseases. Conversely, local injection is clinically feasible for treating periodontitis because the injections can be directly administered into the gingiva. A previous study by our group demonstrated that cells implanted in artificial periodontal defects resulted in the regeneration of periodontium within 2 weeks from cell implantation (19). Furthermore, some studies suggest that MSCs have the ability to engraft damaged tissues, integrate into tubular cells, and differentiate into mesangial cells (28,29). Nevertheless, directly administered BMSCs may exhibit diminished survival by the lack of available oxygen or nutrients in | 3,068,932 | 5662686 | 0 | 16 |
some target tissues (27). In this study, the locally administered DiI-labeled BMSCs were detected at 4 days after cell administration in ligature-induced progression of periodontitis in conjunction with the diminished periodontal bone resorption level, indicating that this method was potent in preventing periodontitis progression. However, further studies are necessary to determine the survival rate of administered BMSCs over a time span of >4 days. Next, we examined the effect of BMSCs dosage on the extent of treatment response. We found that the administration of 10 6 cells was significantly better at suppressing alveolar bone loss than other doses examined. Therefore, this dosage was selected for subsequent analyses. BMSCs were administered 3 days after ligature placement with a single dose into the mesial area of the root because previous studies showed that inflammation arises within 2 days of ligature placement, beginning around the cervical area and eventually leading to a chronic inflammatory response that results in alveolar bone loss (30)(31)(32). The ligature was maintained in the subgingival sulcus for the entire experimental period because its removal facilitated periodontal repair (33). The location of BMSC administration was carefully considered. We administered BMSCs into the mesiobuccal gingiva at the second molar root; however, a greater difference was observed in alveolar bone loss and periodontal integrity between the mesial and distal areas at the second molar root in the ligature + BMSC group, suggesting that the cell microenvironment regulates the effect of locally administered BMSCs. However, further analysis is necessary to completely understand these effects. Our analysis of alveolar BV | 3,068,933 | 5662686 | 0 | 16 |
revealed a significant decrease in the ligature control group vs. the ligature + BMSC group. In particular, the alveolar bone comprising the buccal wall of the second molar roots was clearly recognized in the ligature + BMSC group compared with that in the ligature control group. Moreover, fewer osteoclasts and odontoclasts were present in BMSCadministered mice, suggesting that these cells possibly ameliorated alveolar bone resorption by blocking osteoclast and odontoclast recruitment. Furthermore, BMSC-treated mice showed superior preservation of the principal fiber structure in the gingival and periodontal ligaments, indicating that BMSCs were well distributed over the connective attachment. Within the limitations of this study, the results showed that BMSC administration facilitated periodontal preservation in a mouse model of periodontitis because the administered cells could be effectively distributed in vivo. Despite our findings, the mechanism by which BMSCs mitigate the effects of periodontitis remains unclear. One possibility is that BMSCs differentiate into cementoblasts, osteoblasts, and periodontal fibroblasts in vivo. However, in our study, Dil-labeled BMSCs were observed only in the gingiva, and no supporting evidence exists to suggest that BMSCs can differentiate into these cell types. A more likely possibility is that BMSCs suppress proinflammatory biological mediators because these cells exert profound antiinflammatory and immunomodulatory effects via various mechanisms that are most remarkable for cytokine and chemokine secretion (23,(34)(35)(36). Nevertheless, further studies are warranted to evaluate the effects of immunomodulators on host inflammatory response. MSCs can act through multiple mechanisms to coordinate a dynamic, integrated response to periodontitis, and additional studies to examine these multicellular effects are | 3,068,934 | 5662686 | 0 | 16 |
warranted. The attractive outcomes observed in this study, in addition to the known antiinflammatory functions of BMSCs, support their continued investigation as a potential treatment modality for periodontal diseases. In conclusion, this study demonstrates that BMCs locally applied are able to survive for 4 days during the progression stage of periodontitis and that these cells effectively decreased bone resorption. Although the precise mechanisms underlying BMSCs function remain elusive, a number of antiinflammatory soluble factors, secreted by BMSCs, have already been implicated in other disease models, suggesting that a panel of factors similar to that produced by BMSCs is also responsible for the host protective effects observed in this study, but this remains to be elucidated in future studies. | 3,068,935 | 5662686 | 0 | 16 |
Computerized Human Body Modeling and Work Motion-capturing in a 3-D Virtual Clothing Simulation System for Painting Work Clothes Development By studying 3-D virtual human modeling, motion-capturing and clothing simulation for easier and safer work clothes development, this research aimed (1) to categorize heavy manufacturing work motions; (2) to generate a 3-D virtual male model and establish painting work motions within a 3-D virtual clothing simulation system through computerized body scanning and motion-capturing; and finally (3) to suggest simulated clothing images of painting work clothes developed based on virtual male avatar body measurements by implementing the work motions defined in the 3-D virtual clothing simulation system. For this, a male subject's body was 3-D scanned and also directly measured. The procedures to edit a 3-D virtual model required the total body shape to be 3-D scanned into a digital format, which was revised using 3-D Studio MAX and Maya rendering tools. In addition, heavy industry workers' work motions were observed and recorded by video camera at manufacturing sites and analyzed to categorize the painting work motions. This analysis resulted in 4 categories of motions: standing, bending, kneeling and walking. Besides, each work motion category was divided into more detailed motions according to sub-work posture factors: arm angle, arm direction, elbow bending angle, waist bending angle, waist bending direction and knee bending angle. Finally, the implementation of the painting work motions within the 3-D clothing simulation system presented the virtual painting work clothes images simulated in a dynamic mode. I. Introduction It is reported that there are various | 3,068,936 | 111827747 | 0 | 16 |
hazardous factors to the work environment, that consequently cause the low level of work safety in the primary manufacturing industries in South Korea such as construction and heavy industries (KOSHA, 2012). In the preliminary study (Bae, Park and Park, 2010b), the three major heavy industries in South Korea, i.e. automobile, machine, and shipbuilding were investigated, and the main manufacturing works in these industries were divided into 12 processes, each of which was classified based on the impact indexes of environmental factors and motion factors on the involved workers. The manufacturing process in automobile, machine and shipbuilding industries, which utilized steel as the primary material, included such operations as cutting, molding, assembly, welding, mounting, transferring, painting and inspection of the steel processed goods. In particular, painting was one of the most important processes that prevents the external parts from being corroded during or after the processing and that maintains the outer appearance refined (Bae, Park and Park, 2010a). Such painting work is essential in these industries in reflection of the demands in the business work processes, however most of the pigments used consist of a large amount of additives including organic compounds, and organic solvents contained in thinners for antifouling paints such as benzene, toluene, and xylene. These organic solvents stimulate the worker's olfactory sense during the inhalation of the vapor, which may cause fatigue and dermatitis and dry skin upon pigmentation (Sim, Jeoung, Lim, Lee and Kim, 2009). In addition, long period or short period exposure to such organic compounds may cause dyspnea, headache, suffocation, coma, | 3,068,937 | 111827747 | 0 | 16 |
anxiety, insomnia, prostration, chronic skin disease, and so forth (Kim, Chung, Jeong, Sur and Moon, 1997). Based on the results from the preliminary study (Bae et al., 2010b), it turned out that work processes in automobile, machine and shipbuilding industries involved such harm factors to the work environment as temperature, oxygen deficiency and exposure to harmful chemicals, metal particles and organic solvents. As the level of harms of each environmental factor was similarly high to each other, painting work in both machine and shipbuilding industries was classified as a similar process within these machine, automobile, and shipbuilding industries. Ashdown and Watkins(1996) and Park(2011) highlighted the importance of the consideration of the protective performance from work environmental harm factors as well as the wearer mobility (Rosenblad, 1985) regarding to work clothes development. However, there were particularly certain difficulties to control the evenness of experimental factors, for example, the iteration of work motions designed for the wearer mobility tests, the adjustment of human subject body measurements and experimental clothes sizes and most of all, unnecessary risks to expose the human subject's body to harmful work environmental elements regarding to the wearer mobility evaluation of work clothes prototypes to be developed. To take into account all these difficulties, it seems to be in urgent requirement to conduct researches on 3-D virtual clothing simulation for easier and safer work clothes development as well as its appearance and wearer mobility tests. Therefore, this research aimed first, to categorize painting work motions by video recording painting Modeling To generate a 3-D virtual | 3,068,938 | 111827747 | 0 | 16 |
human male model, a human male subject was 3-D scanned and also directly measured in terms of body measurement items, that were height, bust circumference, waist circumference, hip circumference, shoulder length, neck width, back across width, arm length, waist back length, body rise, crotch height, knee height. The procedure to edit a 3-D virtual male model required the total body shape 3 dimensionally scanned in a digital format, which was revised by using 3D Studio MAX(3DS Max®, 2015) and Maya (Maya®, 2015), 3-D modeling and rendering tools. Computerization of Painting Work Motion-capturing Based on the painting work posture characteristics defined ahead, a human male subject was asked to perform 9 painting work motions following motion-capturing procedures (refer to figure 3). The painting work motion data collected were, in turn computerized to establish virtual work motions within a 3-D clothing simulation system. Characteristics of the 3-D Virtual Male Model Avatar To scan the whole body shape and to capture the motions of the subject in a digital format, the subject wore a fitted suit marked with sensors to perceive major body measurement points for 3-D body scanning, which is shown in Figure 3. The scanned data of body and work motions were collected into the computer and consequently frame edited and surface rendered (refer to Figure 4) through 3D Studio Max® and Maya® software(AutoDesk Inc., 2015). Figure 5 presents the completed male avatar's body with the skin and hair texture in the views of front, side and back. Afterward, the 3-D clothing simulation system would apply the | 3,068,939 | 111827747 | 0 | 16 |
9 painting work motions defined in the study to this virtual avatar model developed for the painting work clothes simulation. Painting Work Motion-capturing To motion-capture the painting work motions in a digital format, 9 painting work motions were defined for the study. waist bending direction factor: front/side direction; knee bending angle factor: 45°/90°/180° bending range. Also, photos about motion-capturing these work motion details are shown in Table 3. The 3-D male avatar performed these 9 painting work motions within the 3-D virtual clothing simulator, DC Suite, which are shown in Figure 6 according to each painting work motion category. ❚A human subject in a 3-D scanning suit ❚A human subject performing work motions to be scanned in a digital format The jumper and pants type of painting work clothes patterns were drafted based on the men's work jumper and pants block pattern -making methods (Park and Lee, 2012;Park, 2013). Figure 7 shows the work clothes patterns worn by the male avatar within the 3-D clothing simulation system. In conclusion, the 3-D painting work jumper and pants clothing simulation finally produced the virtual images worn by the virtual male avatar while performing given work motions in a dynamic simulation mode (refer to Figure 8). Which means the usage of the virtual clothing simulator can be extended from dealing with the fashionable items in the context of the appearance evaluation to the industrial clothes and equipments for the investigation of the wearer's iterative movement without fatigue and excluding the harmful environmental factors. IV. Conclusion and Suggestion The results | 3,068,940 | 111827747 | 0 | 16 |
derived from the research highlighted that the work motions observed at manufacturing sites were varied in multiple categories and wide posture factor ranges. Concerning the research aims, the research has been successful to define the painting work motions and consequently to implement the digital format data of a human male subject's body features and painting work motions captured to the 3-D clothing simulation system for the study. It also effectively suggested the virtual images of the painting work clothes 3-D In particular, the 3-D painting work clothes simulated images were caught in various aspects including cross-sectional views of the clothes, that would evaluate its appearance effect and the wearer mobility by analyzing percentage of voids between body and clothes at the level of selection (refer to Figure 9). | 3,068,941 | 111827747 | 0 | 16 |
Shelf-life extension of wheat flour by irradiation technique Irradiation is the process of exposing the food to some dose of radiation to extend the shelf life of the food. The present study was done on shelf-life extension of wheat four by gamma radiation. Gamma rays are used for food processing, whose light source is obtained from a 60 Co radionuclide source. Irradiation properties of gamma rays have adverse effects on nutritional properties of whole wheat flour with dose less than 5 kGy. The physiochemical properties and the nutritional properties of wheat has been analysed. The moisture content of untreated flours (8.69%), treated flour (7.73%) and microbial activity of untreated flour of the whole wheat flour has been reduced by the dose of radiation. However, the physiochemical parameters of wheat flour such as, ash untreated (1.98%) treated (1.93%), fat untreated (1.75%) treated (1.74%), protein untreated (16%) treated (9.27%), fibre untreated (2.06%) treated (2.10%), has been analysed when introduced to radiation. The techno-functional parameters of treated and untreated wheat flour such as, water absorption capacity (WAC), water absorption index (WAI), bulk density has no effect while introduced to radiation. The effect of irradiation on food has been studied that at high dose of radiation, there is more liable to flavour change. Introduction Cereals are very important crops from the economic, agronomic, and consumer point of view. Among cereals, rice and wheat are in high demand and are cultivated on a large scale. Wheat is one of the most important staple foods around the world. The presence of nutrients | 3,068,942 | 259928858 | 0 | 16 |
such as starch, protein, minerals, vitamins, and lipids makes wheat flour highly promotable as a balanced diet (Xiao et al., 2020) [1] . Wheat and its by-products are utilized in a wide range, wheat is mainly used as raw material in bakery products, beverage industry, cosmetics, ethanol production, and also as animal feed. Whole wheat flour consumption protects humans against diseases such as constipation, appendicitis, non-alcoholic fatty liver diseases, and obesity (Kumar et al., 2011) [22] . Wheat is one of the world most commonly consumed cereal grain. It comes from a type of grass (Triticum) that is grown in countless varieties worldwide, white and whole-wheat flour are the key ingredients in baked goods, such as bread. Other wheatbased foods include pasta, noodles, semolina, bulgur, and couscous (Ahmad et al., 2014). Wheat is mainly composed of carbohydrates, but also has a moderate amount of protein. (Calories-340, Protein-13.2gm, carbohydrates-72 gm, fiber-10.7 gm, fat-2.5 gm). starch is the predominant carbohydrate in the plant kingdom, accounting for over 90% of the total carbohydrate content in wheat. wheat is mainly composed of carbohydrates, fibre content in of whole wheat grain is 12-15% of the dry weight. As they are concentrated in the bran, fibre is removed during the milling process and largely absent from refined flour. The main fibre in wheat bran is arabinoxylan (70%), which is a type of hemicellulose. The rest is mostly made up of cellulose. Vitamins and minerals, as with most cereals grains (Erkmen et al., 2016) [23] . The amount of minerals depends on the | 3,068,943 | 259928858 | 0 | 16 |
soil in which its grown. Selenium this trace element has various essential functions in our body. Manganese is found in high amounts in whole grains, manganese may be poorly absorbed from whole wheat due to its phytic acid content. Phosphorus this dietary mineral plays an essential role in the maintenance and growth of body tissues (Kirwan et al., 2013) [24] . The main concern of agriculture is pre and post-harvest losses, the significant losses are caused by inadequate storage conditions as well as decisions made at earlier stages of the supply chain, including transportation, storage, and processing, which predispose products to a shorter shelf life. The true extent of post-harvest losses is the subject of some dispute as they are difficult to measure accurately. According to the world food programme, 40% is common. In Africa, post-harvest losses of maize from harvest sales are believed to amount to around 10-20%, Approximately 40% of these losses occur during storage at the farm and market, 30% during processing (drying, threshing, and winnowing), 20% in transport from the field to the home stand, and the remaining 10% during transport to market. As there is high chances of losses during storage (i.e 40%) of cereals. extending the shelf life of food products is one of the main objectives of food producers, especially about the perishable foods. One of the most important methods for extending of shelf life is by irradiation treatment, which will ultimately improve food health. The awareness in consumers about the health benefits associated with the consumption of whole | 3,068,944 | 259928858 | 0 | 16 |
grain foods has increased their demand to a large extent. Whole wheat flour is a rich source of antioxidants which play an important role in preventing cancer and cardiovascular diseases. However, the shelf life of wheat grains is limited to 6-8 weeks owing to insect infestation even in sealed pouches. The conventional methods of fumigation are not possible for sealed pouches as the fumigants cannot penetrate the same. Therefore, gamma irradiation provides an alternative method for preventing insect infestation as well as food spoilage and food-borne illness (Salem et al., 2016) [25] . The main concern of agriculture is pre and post-harvest losses, the significant losses are caused by inadequate storage conditions as well as decisions made at earlier stages of the supply chain, including transportation, storage, and processing, which predispose products to a shorter shelf life. The true extent of post-harvest losses is the subject of some dispute as they are difficult to measure accurately. Early experiments showed that ionizing radiation kills bacteria. Promising and scientifically interesting as they were, these early efforts did not lead to the use of ionizing radiation by the food industry. At the turn of the century and for many years thereafter, there was no costeffective way of obtaining radiation sources in the quantity required for industrial application. The X-ray generators of the day were very inefficient in converting electric power to X-rays, and the naturally occurring radioactive materials, such as radium, were too scarce to provide gamma rays, or other forms of radiation, in sufficient quantities for food processing. | 3,068,945 | 259928858 | 0 | 16 |
To overcome these losses modern techniques like irradiation treatments are used to increase the shelf of the product. Food irradiation is one of the modern, secure, and efficient ways for its preservation, which is practised in many countries around the world (Bason et al., 2017) [5] . Food irradiation has an advantage over traditional methods as it is likely to maintain food quality for longer periods. Food irradiation is a very mild treatment, as absorption of radiation dose of 1 KGY is able to increase the temperature of the product by 0.36 °C. This indicates the minimum nutritional losses in irradiation than heating, drying and cooking. Along with that irradiated foods do not initiate the production of heterocyclic ring compounds and carcinogenic aromatics as it is found in thermal processing of food at high temperatures (Bornare et al., 2018) [26] . Irradiation techniques like gamma, electron beam, and X-ray are used for food irradiation. Among them, gamma irradiation is more popular for food processing and food irradiation because of its high penetration power and efficiency to eliminate contamination. Also, electron beam irradiation is not preferred over gamma irradiation because electron beam irradiation induces the formation of free radicals such as hydroxyl radicals and peroxyl radicals (Brasoveanu et al., 2013) [27] . Food irradiation, (the application of ionizing radiation to food) is a technology that improves the safety and extends the shelf life of foods by reducing or eliminating microorganisms and insects. Like pasteurizing milk and canning fruits and vegetables, irradiation can make food safer for the | 3,068,946 | 259928858 | 0 | 16 |
consumer. The Food and Drug Administration (FDA) is responsible for regulating the sources of radiation that are used to irradiate food. The FDA approves a source of radiation for use on foods only after it has determined that irradiating the food is safe (Kulsum et al., 2020) [2] . Gamma rays are produced from radioisotopes cobalt-60 and cesium-137, cobalt-60 is produced in a nuclear reactor via neutron bombardment of highly refined cobalt-59 pellets, while cesium-137 is produced as a result of uranium fission, both cobalt-60 and cesium-137 emit highly penetrating gamma rays. Radiated food prevents of food borne illness-to effectively eliminate organisms that cause food-borne illness, such as salmonella and Escherichia coli. Prevention to destroy or inactive organisms that cause spoilage and decomposition and extend the shelf life of foods. Control of insects-to destroy insects in or on tropical fruits imported into the United States. Irradiation also decreases the need for other pest-control practices that may harm the fruit. Delay of sprouting and ripening to inhibit sprouting and delay ripening of fruit to increase longevity (Haripriya et al., 2010) [3] . The gamma irradiation studies reported changes in wheat flour protein if irradiated above 10 KGY dose. Modification in wheat protein leads to a decline in dough property, which affects the quality and life of bakery products like bread and buns. The effect of gamma-ray was reported on a few properties of wheat flour at particular doses like 0.5, 1, 2.5, 5, and 10 kGy. Satin et al., (2002) [4] studied the effect of gamma irradiation | 3,068,947 | 259928858 | 0 | 16 |
on the physicochemical properties of whole wheat flour samples at 2.5 and 5 kGy doses. Their results revealed a significant decrease in water absorption, oil absorption, swelling power and emulsion capacity whereas, water solubility index, emulsion stability, foaming capacity and stability were found to increase upon irradiation. Decrease in pasting properties and intensities of few bonds in the structural analysis were reported with irradiation. On the contrary, no changes in the proximate composition, bulk density and FTIR spectra pattern were reported with dosage. Also, a significant increase in amylose content, swelling, solubility, syneresis and freeze-thaw stability, water and absorption capacity of the flour with dosage was reported by (Bashir et al., 2017) [28] . The research group of Khan et al., (2018) [7] reported no change in the chemical composition of wheat flour and increasing dough water absorption in wheat flour irradiated at 5 kGy. The literature reported so far gives mixed results on the effects of gamma irradiation on proximate composition, bulk density, functional properties and FTIR spectra of wheat flour. Satin et al. (2002) [4] observed that the food irradiation is a process exposing food to ionising radiations such as gamma rays emitted from the radioisotopes 60 Co and 137Cs, or, high energy electrons and X-rays produced by machine sources. Depending on the absorbed radiation dose, various effects can be achieved resulting in reduced storage losses, extended shelf life and/or improved microbiological and parasitological safety of foods. However, hindering factors in the way of commercial implementation of the food irradiation process are politics and | 3,068,948 | 259928858 | 0 | 16 |
consumer advocacy. A similar situation occurred with the heat pasteurisation of milk in the past. ~ 729 ~ The Pharma Innovation Journal https://www.thepharmajournal.com Moy et al. (2002) [29] stated that the potential application of ionising radiation in food processing is based mainly on the fact that ionising radiations damage very effectively the DNA so that living cells become inactivated, therefore microorganisms, insect gametes, and plant meristems are prevented from reproducing, resulting in various preservative effects as a function of the absorbed radiation dose. At the same time, radiation-induced other chemical changes in food are minimal. According to the Codex General Standard for Irradiated Foods CAC. (2003), it has been reported that ionising radiations foreseen for food processing are limited to high energy photons (Gamma rays of radionuclide's 60 Co and, to a much smaller extent, 137 Cs, or, X-rays from machine sources with energies up to 5 MeV, or accelerated electrons with energies up to 10 MeV. In the USA, the Food and Drug Administration amended recently the food additive regulations by establishing a new maximum permitted energy level of X-rays for treating food of 7.5 MeV provided that the X-rays are generated from machine sources that use tantalum or gold as the target material. High-energy electron beams are produced by electron-accelerating machines. X-ray production starts with high-energy electrons: X-ray machines convert electron energy to electromagnetic X-rays called "Bremsstrahlung" These types of radiation are chosen because they produce the desired food preservative effects, they do not induce radioactivity in foods or packaging materials, they are available | 3,068,949 | 259928858 | 0 | 16 |
in quantities and at costs that allow commercial use of the irradiation process. Bradley et al. (2015) [30] examined differences in radiation sensitivities among the microorganisms are related to differences in their chemical and physical structure, and in their ability to recover from radiation injury. The amount of radiation energy required to control microorganisms in food, therefore, varies according to the resistance of the particular species and according to the number of organisms present. Besides such inherent abilities, several factors such as the composition of the medium, the moisture content, the temperature during irradiation, the presence or absence of oxygen, the fresh or frozen state influence radiation resistance, particularly in the case of vegetative cells. Similar to heat resistance, the radiation response in microbial populations can be expressed by the decimal reduction dose. Summarizing data from a large number of references, presents typical radiation resistances of a number of bacteria in nonfrozen foods. The radiation sensitivity of many moulds is of the same order of magnitude as that of vegetative bacteria. However, fungi with melanised hyphae have a radiation resistance comparable to that of bacterial spores Yeasts are as resistant as the more resistant bacteria. Viruses are highly radiation resistant. Basson et al. (2017) [5] observed early experiments that showed that ionizing radiation deteriorates bacteria. Promising and scientifically interesting as they were, these early efforts did not lead to the use of ionizing radiation by the food industry. At the turn of the century and for many years thereafter, there was no cost-effective way of obtaining | 3,068,950 | 259928858 | 0 | 16 |
radiation sources in the quantity required for industrial application. The X-ray generators of the day were very inefficient in converting electric power to X-rays, and the naturally occurring radioactive materials, such as radium, were too scarce to provide gamma rays, or other forms of radiation, in sufficient quantities for food processing. Khan et al. (2018) [7] studied irradiation food preservation uses high energy, known as ionizing radiation. It is because the material in its path can be ionized. When irradiation sources such as X-rays, gamma rays, and electron beams touch the material, these foods, and components will be excited, ionized, and altered. An excitation is an event where living cells become sensitive to external conditions. Ionization is the process by which macromolecules are broken into free radicals. Changes in living cells components will inhibit DNA synthesis, disrupting microbial cell division, and biological effects. This effect inhibits microbial growth in food. Rawat et al. (2015) [8] reviewed gamma rays are used for food processing, whose light source is obtained from a 60 Co radionuclide source. This type of radiation is essentially monoenergetic. Using analytical methods such as the kernel point or the Monte Carlo method, it is straightforward to calculate the spread of irradiation doses in food products. The resulting dose depth distribution will resemble an exponential curve. Irradiation from two sides, obtained by rotating the processing load, is often used to enhance dose uniformity in system loads. Its applications on food were widely used. Kim et al. (2004) [31] reported gamma rays, X-rays, visible light, and | 3,068,951 | 259928858 | 0 | 16 |
UV are all of electromagnetic (EM) radiation. EM radiation differs in frequency and hence in energy. Gamma rays are the most energetic form of such electromagnetic radiation, having the energy level from around 10 keV to several hundred keV volts, and therefore they are more penetrating than other radiation such as alpha and beta rays. Gamma rays belong to ionizing radiation and interact to atoms or molecules to produce free radicals in cells. These radicals can damage or modify important components of plant cells and have been reported to affect differentially the morphology, anatomy, biochemistry, and physiology of plants depending on the irradiation level. These effects include changes in the plant cellular structure and metabolism, e.g., dilation of thylakoid membranes, alteration in photosynthesis, modulation of the ant oxidative system, and accumulation of phenolic compounds. Barbara et al. (2002) [9] studied physiological symptoms in a large range of plants exposed to gamma rays have been described by many researchers. The symptoms frequently observed in the low-or high-dose-irradiated plants are enhancement or inhibition of germination, seedling growth, and other biological responses. The growth of Arabidopsis seedlings exposed to low dose gamma rays (1 or 2 kGy) was slightly. The relationship between growth of irradiated plants and dose of gamma irradiation has been manifested by investigating the morphological changes and seedling growth of the irradiated plants. No significant morphological aberrations were observed in the phenotype of the plants irradiated with relatively low doses (1-5 kGy) of gamma rays, while a high-dose (50 kGy) irradiation inhibited seedling growth remarkably. Sources | 3,068,952 | 259928858 | 0 | 16 |
of irradiation Rawat et al., (2015) [8] stated that irradiation an essential requirement for the industrial use of food irradiation is an economic source of radiation energy. Two types of radiation source can satisfy this requirement today: machines and manmade materials. Although they differ in the method of operation, both types of source produce identical effects on foods, microorganisms, and insects. Machines called electron accelerators produce electron radiation, a form of ionizing The Pharma Innovation Journal https://www.thepharmajournal.com radiation. Electrons are sub-atomic particles having very small mass and a negative electric charge. Beams of accelerated electrons can be used to irradiate foods at a relatively low cost. this cost advantage is offset, however, by the fact that accelerated electron beams can penetrate food only to a maximum depth of about 8 em, which is not deep enough to meet all the goals of food irradiation. Accelerated electrons are, therefore, particularly useful for treating grain or animal feed that can be processed in thin layers; electron beam irradiation is particularly suitable for these applications because of the very high throughputs involved in grain handling. Charlesby et al. (2009) [16] studied man-made radionuclides constitute the other main source of ionizing radiation; radionuclides are radioactive materials that, as they decay give off ionizing gamma rays that can be used for food processing. One radionuclide that is readily available in large quantities is cobalt-60, which is produced by exposing naturally occurring cobalt-59 to neutrons in a nuclear reactor. The availability of another radionuclide, caesium137, a byproduct of nuclear reactor operations, is | 3,068,953 | 259928858 | 0 | 16 |
limited and it is not used widely at present. Gamma rays from either of these radionuclides will penetrate deeply enough to meet virtually all food irradiation needs. The cost of man-made radionuclide sources is considered acceptable for industrial food irradiation in view of the great versatility and penetrating capacity of the gamma-rays. Mustafa et al. (2013) [10] analysed low doses of irradiation can be applied to several types of food products such as tubers, fresh fruit and vegetables, cereals or nuts and seeds, dried vegetables and spices, and dry food of animal origin. Irradiation aims to delay ripeness, eradicate insects, and control quarantine for some fresh fruits and vegetables. Irradiation of tubers seeks to inhibit the germination process. Doses of irradiation and studies Low dose of irradiation In cereals or nuts and seeds aims to eliminate insects. Meanwhile, irradiation of dry vegetables, spices, or dry herbs and tea herbs is intended to eradicate insects. Dry food derived from animals also has the same goal, namely, to eliminate insects. Recommendations or regulations regarding irradiation in low doses of food products are available based on the BPOM Republic of Indonesia. The minimum dose is 0.2; 1.0; 1.0; 1.0; and 1.0 kGy, respectively found in tubers; fresh vegetables and fruits; cereals, beans and seeds; dry vegetables and spices; and dry food from animal sources High dose of irradiation Wojcik et al. (2019) [11] reported that high levels of irradiation can be applied to food items, such as dried spices and animalbased ready-to-eat food. Each irradiation process that is carried | 3,068,954 | 259928858 | 0 | 16 |
out has a different purpose. Irradiation of some dried herbs has the aim of reducing certain pathogenic microorganisms. Meanwhile, animal-based ready-to-eat processed food products have the objective of sterilizing and eradicating pathogenic microbes, including microbes with spores and extending shelf life. There are regulations regarding irradiation in high doses of food, including some dry spices with a minimum dose of 10.0 kGy. Ehlermann et al. (2016) [12] studied irradiation can change the chemical compounds in food to change these products' nutritional value in nutritional aspects. The study results showed that nutrient loss in food irradiated with 1 kGy dose had no significant impact. In comparison, moderate-dose irradiation (1-10 kGy) may reduce its nutritional components unless the irradiation process's temperature and air are adjusted in this way. Proper treatment, such as combined radiation conditions with packaging techniques, will maintain processed food products' quality and nutrition. Younis et al. (2020) [13] examined the benefits of using irradiation in food are little or no heating process, so the material doesn't change its characteristics. Also, irradiation can suppress microorganisms that live in food. Irradiation can be carried out on packaged foods, frozen foods, and fresh food through one operation and do not use chemical additives. Irradiation requires only a small amount of energy, nutrition changes can be compared with other preservation methods, the automatic process is controlled, and the operating costs are low. Zhao et al. (2017) [37] reported that irradiation food preservation uses high energy, known as ionizing radiation. It is because the material in its path can be | 3,068,955 | 259928858 | 0 | 16 |
ionized. When irradiation sources such as X-rays, gamma rays, and electron beams touch the material, these food components will be excited, ionized, and altered. An excitation is an event where living cells become sensitive to external conditions. Ionization is the process by which macromolecules are broken into free radicals. Changes in living cell components will inhibit DNA synthesis, disrupting microbial cell division, and biological effects. This effect inhibits microbial growth in food. Some of the benefits of using irradiation in food are little or no heating process, so the material doesn't change its characteristics. Also, irradiation can suppress microorganisms that live in food. Irradiation can be carried out on packaged foods, frozen foods, and fresh food through one operation and do not use chemical additives. Irradiation requires only a small amount of energy, nutrition changes can be compared with other preservation methods, the automatic process is controlled, and the operating costs are low. Sood et al. (2020) [13] observed that each food ingredient irradiated with a different purpose has an extra dose depending on the food itself characteristics. The rays commonly used to preserve food products with irradiation are gamma rays, X-rays, and electron beams, each of which has its advantages and disadvantages. Chemical, nutritional, microbiological, and toxicological aspects are used for food irradiation safety parameters. The principle of radiation is excitation, ionization, and changes in components contained in foods when the radiation source touches the material. The irradiation process has advantages and disadvantages, one of which is that the radiation process does not use heat | 3,068,956 | 259928858 | 0 | 16 |
so food does not change its characteristics. However, the irradiation process is still a public fear of radioactive influence on foodstuffs. Irradiation can be applied to foods by paying attention to the dose according to the foodstuff. Effect of food irradiation Urbain et al. (2012) [32 ] stated that at high energy levels, ionizing radiation can make certain constituents of the food radioactive. Below a certain threshold of energy, however, these reactions do not occur. On the basis of experimental studies and theoretical estimates, in 1980, the Joint FAO/IAEA/WHO Expert Committee on the Wholesomeness of Irradiated Foods recommended restricting the radiation sources used in food processing to those with energy levels The Pharma Innovation Journal https://www.thepharmajournal.com well below those that induce radioactivity in treated food. Food processed by radiation in accordance with the Committee's recommendations does not become radioactive. However, the chemical composition of food can be altered by radiation, and authorities responsible for assessing the safety of irradiated food have had to consider the possibility that some of the chemical compounds formed during food irradiation may be harmful. In recent years, radiation chemistry has been recognized as an additional tool for toxicological evaluation, and the methods involved have been substantially refined. As a result, answers to questions about the safety of irradiated food can be extrapolated with reasonable confidence on the basis of information about the chemical composition of foods and the radiolytic effects (Chemical changes caused by irradiation) produced under various conditions. Effect of irradiation on mechanical properties of grain Padmashree et al. (2016) | 3,068,957 | 259928858 | 0 | 16 |
[14] reported that IR radiationinduced remarkable changes in the mechanical and physical properties of the grain. It resulted in a decrease in elasticity modulus, bio-yield point and the compression resistance resulting in an increase in stiffness, and breakage susceptibility as studied in case of barley, lentils, kidney bean, black bean and pinto bean. These changes were attributed either to the starch gelatinization during heating or to the fissuring of the grain surface. In case of buckwheat, IR radiation reduced the bulk density which was attributed to the diffusion of water from inside to outside, resulting in volume expansion. The exposure of wheat grain with IR radiation increased the kernel hardness which was attributed to the interaction between starch and protein. The removal of moisture during IR heating might have exposed the free atoms in both starch and protein subsequently facilitating their interaction. In case of rice, the IR heating increased milling yield due to the uniform heating and less moisture gradient. Apart from the uniform heating mechanism of IR radiation, the phenomenon of increased hardness might also have contributed to the improved milling yield. Sciarini et al. (2015) [15] reported that IR treatment on wheat grain can significantly affect particle size of the bran and flour, thus improving their functional properties. Similarly, IR irradiated lentil flour particles were of smaller size compared to non-irradiated. Author also observed that the particle size of the lentil flour irradiated with IR at lower temperature and higher moisture content had larger particle size due to clumping of the particles. Hence, | 3,068,958 | 259928858 | 0 | 16 |
IR thermal treatment might be considered as an alternative green technology to reduce the flour particle size, where moisture content is a critical factor. Although the reduction in particle size could also be achieved by regrinding, it caused a higher degree of starch damage and affected final product quality. The IR radiation cause physicochemical changes in grain starch, such as changes in moisture content, solubility, and swelling ability. The extent of the aforementioned changes depends on the starch type, moisture content, exposure time, processing temperature and absorption of IR thermal energy. Sukalovic et al. (2010) [17] stated that the IR radiation interacts with grain and its components in an interesting way by vibrating the bonds causing it to stretch, unlike the conventional thermal treatment and has an ability to modify characteristic properties of the starch. Since, starch is the major component of grain, any change in the above observed properties of starch in turn changes the mechanical and functional properties of the grain, and therefore starch characteristics defines the final application in the food industry. The IR treatment of grains reduces the energy requirement during flour milling due to the reduced compression resistance, rupture force, modulus of toughness and elasticity. This resulted in an increase in the milling yield and hence, reducing the cost input in the flour milling industry. The additional feature of IR radiation is the ability to modify the functional properties of the grains/flours/starches, and influences the end use. IR irradiated starches had increased final viscosity, water absorption capacity, and water swelling indices, | 3,068,959 | 259928858 | 0 | 16 |
such modified starches can find applications in the formulations such as puddings, soups, dressings, gravies and sauces. Rastogi et al. (2015) [18] reviewed the properties like extensibility, viscosity, etc. are considered as a key macroscopic property in the formation and development of food products. One of the factors affecting rheological properties is thermal energy. Several studies have shown that the IR irradiation caused a reduction in the hot paste consistency and in peak and breakdown viscosities as observed in wheat and corn starch. The decrease in breakdown viscosity was attributed to the reduced ability of starch granules to swell and to the reduction in the proportion of long amylopectin chains during treatment. An increase in cold paste viscosity was also noticed and it was attributed to the increased resistance of starch granules to the breakage. Another study on rice revealed that the annealing of starch and denaturation of protein could reduce the peak and final viscosity. Similar changes in the pasting properties of maize, lentils, and buckwheat were also reported. The IR treatment also reported to decrease set back and final viscosity in mung bean, which was attributed to the inability of disrupted starch to realign itself during retrogradation. Yet another study on IR treated maize reported similar reductions in final viscosity of IR-treated maize due to changes in the starch structure and solubility of the protein. The IR radiation-induced changes in the wheat starch, and protein which in turn reduced the values of farinograph, mixogram, and extension graph for the wheat dough, which could be | 3,068,960 | 259928858 | 0 | 16 |
disadvantageous in the bakery industry as it reduces the dough stability and extensibility. Ding et al. (2016) [19] studied the IR radiation-induced changes in the wheat starch, and protein which in turn reduced the values of farinograph, mixogram, and extension graph for the wheat dough, which could be disadvantageous in the bakery industry as it reduces the dough stability and extensibility. However, a reduction in dough development time was reported. The above studies clearly demonstrated that the disintegration of starch granules causes the reduction in values of pasting profile in all the IR treated grains and starches, except waxy varieties of barley indicating the importance of amylose in deciding the rheological properties. In addition to starch, the protein content also seemed to affect the rheological properties which might be the reason for the difference in the behaviour of monocots and dicots to IR radiations. These changes in the rheological properties of starches and grains have implications in their final application in the food industry. Also, the insight of these changes by IR radiation might assist in optimizing processing parameters such as time, moisture content and energy input. IR radiation caused an increase in peak, setback, breakdown, and final viscosity of the tempered and non-tempered waxy variety of Effect of irradiation on functional properties of grain The Pharma Innovation Journal https://www.thepharmajournal.com barley, whereas a reduction in breakdown viscosity of normal barley commensurate with the increase in pasting temperature. The author opined that the increase in viscosity of waxy barley is due to the increase in swelling ability | 3,068,961 | 259928858 | 0 | 16 |
of starch, whereas in case of normal barley, starch becomes resistant to lysis and remain stable at higher temperature. However, the interaction between the starch and protein could also play an important role in modifying the swelling ability of starch. Josephson et al. (2015) [20] reported that the high radiation dose required for sterilization has been associated with unwanted flavour changes in some food, and it appears that the change occurs in the lean rather than the fat portion of some food. it decreases or disappears during storage and cooking. Investigators have also observed that particle of food irradiated at low temperature is less liable to flavour change. Enzyme-inactivated food products that received about 50 kGy of radiation at a temperature of -30 °C for long-term shelfstability. The chemical changes that radiation produces in food may lead to noticeable effects on flavour. The extent of these effects depends principally on the type of food being irradiated, on the radiation dose, and on various other factors, such as temperature, during radiation processing. Some foods react unfavourably even to low doses of radiation. Milk and certain other dairy products are among the most radiation sensitive foods. Doses as low as 0.1 kGy will impart an offflavour to milk that most consumers find unacceptable. Wojick et al., (2019) [11] studied that food processing and preparation methods in general tend to result in some loss of nutrients. As in other chemical reactions produced by irradiation, nutritional changes are primarily related to dose. The composition of the food and other factors, | 3,068,962 | 259928858 | 0 | 16 |
such as temperature and the presence or absence of air, also influence nutrient loss. At low doses, up to 1 kGy, the loss of nutrients from food is insignificant. In the medium dose range, 1-10 kGy, some vitamin loss may occur in food exposed to air during irradiation or storage. At high dosages, 10-50 kGy, vitamin loss can be mitigated by protective measuresirradiation at low temperatures and exclusion of air during processing and storage. The use of these measures can hold the vitamin loss associated with high dosage to the levels seen with medium-range doses when protective measures are not employed. Some vitamins-riboflavin, niacin, and vitamin Dare fairly insensitive to irradiation. Others, such as vitamins A, B, E, and K are more easily destroyed. Little is known about the defect of irradiation on folic acid, and conflicting results have been reported concerning the effects of irradiation on vitamin C in fruits and vegetables. (Charlesby et al., 2009) [16] examined the significance of radiation-induced vitamin loss in a particular food depends, of course, on how important that food is as a source of vitamins for the people who consume it. For example, if a specific food product is the sole dietary source of vitamin A for a given population, then radiation processing of that particular food may be inadvisable because it could greatly reduce the availability of this essential nutrient. Furthermore, since many irradiated foods are cooked before use, the cumulative loss of vitamins through processing and cooking should be taken into account. Chemical analyses and animal | 3,068,963 | 259928858 | 0 | 16 |
feeding studies have shown that the nutritional value of proteins is little affected by irradiation, even at high doses. Animal studies in various species have also demonstrated that the effects of radiation on other nutrients are minimal. Effect of irradiation on sensory/nutritional properties Effect of irradiation on gluten and protein content of wheat Sapirstein et al. (2017) [33] studied the influence of gamma irradiation on the rheological behaviour of the dough's obtained from the three wheat cultivars with a micromixograph. The corresponding effects of irradiation on the composition of 50 PI glutenin were assessed by both SDS-PAGE and quantitatively by RP-HPLC. Mixograph peak mixing times and work input to peak values significantly decreased (p<0"05) for all cultivars as irradiation level increased. A similar decrease in peak dough resistance and band envelope area values were also observed with increasing radiation exposure. However, the decrease was significant only for the bread wheat cultivars at the 10 and 20 KGY levels. Significant reductions in peak bandwidth were evident at 10 and 20 KGY irradiation levels for all wheat cultivars. The high, and mostly significant, negative correlations between irradiation dosage and mixograph parameter for all cultivar samples underscores the adverse effect of irradiation on dough mixing characteristics. It has previously been reported that rheological properties of dough's change when bread wheat 4'6 or durum wheat are irradiated. In the present study, irradiation resulted in a weakening effect on the mixing properties, which was more severe at 10 KGY or higher. These findings confirm that wheat gluten has been affected.2 g | 3,068,964 | 259928858 | 0 | 16 |
direct drive mixograph was utilised for the first time for the evaluation of irradiated wheat samples. Because of the computed parameters, it was possible to show the effects of irradiation on rheological properties more clearly than by using the standard mixograph. When this result was explored further, it was found that compared to non-irradiated wheat, the relative decline in total insoluble glutenin at the 20 kGy dosage level ranged from 34% (Be-zostaya) to 47 and 49% (Gerek and Kunduru) respectively. Johnson et al. (2011) [34] observed that gamma-irradiation at the levels investigated had no effect on proteins. In contrast to the gliadins, a significant effect of wheat irradiation was observed in SDS-PAGE patterns of reduced 50 PI glutenin of all cultivar samples; the effect was particularly noticeable at the 10 and 20 kGy dosage levels for the HMW-GS and LMW-GS whose relative intensities decreased considerably compared to control or 2"5-5"0 kGy irradiated samples. It is worth pointing out that the observed decrease in band intensities of HMW-and LMW-GS was more apparent in the original gels than in the corresponding photo-graphs. Quantitative RP-HPLC results confirmed that the concentration of all subunits of the 50 PIglutenin fraction were similarly affected by the irradiation treatments. These results clearly showed that the total peak area of the 50PI glutenin chromatograms decreased significantly (P<0-05 for all wheat cultivars as irradiation level increased. Similar significant decreases were observed for HMW-and LMW-GS. As was observed for mixograph parameters, correlations between irradiation level and 50 PI glutenin composition parameters were high, negative, and generally | 3,068,965 | 259928858 | 0 | 16 |
statistically significant. Pan et al. (2011) [35] studied the thermal treatment influences the basic structure of the starch, which in turn affects its digestibility pattern. There are several studies on cereals and legumes to understand the effect of IR radiation on in vitro starch digestibility. The exposure of IR radiation on normal, high-amylose and waxy barley increased starch digestibility. ~ 733 ~ The Pharma Innovation Journal https://www.thepharmajournal.com At the same time, IR irradiated waxy barley samples contain substantially less amount of resistant starch (RS), The IR radiation had a similar effect on the starch digestibility pattern in cereals, while variations were observed in the case of legumes which might be due to the distribution of starch and protein within the grain. Pulses contain more protein (20-40%) than cereals (1012%). Therefore, protein bodies surrounding starch granules might be the reason for the lesser impact of IR radiation on starch digestibility. The significance of particle size, while assessing the impact of radiation, could be accounted for the differences in the digestibility pattern insitu and in vitro. The difference in in vivo and in vitro studies yielded opposite results suggesting the limitation of the in vitro analysis in mimicking the biological system and its complexity. Therefore, to arrive at a concrete conclusion on the effect of IR radiation on starch digestibility, more scientific insights based on clinical studies are required. Birefringence for native starch granules exhibits a characteristic "maltese cross" at the centre of granules that can be visualized in polarized light. The wheat starch treated with IR radiation | 3,068,966 | 259928858 | 0 | 16 |
at 100 C for 10 and 20 sec did not show any change in the maltese cross. The wheatstarch treated at 750 W power at 30% moisture for 90 min ruptured the granules with black spots at the centre. Bualuang et al. (2013) [38] reported that IR is capable of altering the mechanical and functional properties and starch digestibility pattern of the grain, due to the changes in the structure of starch. The starch granules are spherical or polygonal in shape and exhibit a characteristic birefringence and a diffraction pattern when exposed to polarized light and X-rays respectively. Any change in the birefringence or diffraction indicates the change in the crystalline and amorphous structure of starch. The IR treatment of soft wheat decreased the degree of crystallinity of starch by approximately 26%. Another study showed that IR irradiation of wheat starch at 730 W with 30% moisture for 60 and 90 min changed its native A-type diffraction pattern to V-type with a decrease in relative crystallinity which was attributed to the transformation of the crystalline region to an amorphous region. This transformation in starch was linked to the complex formation of amylose with fatty acids and phospholipids with a V-crystal packing resulting in the V-type diffraction pattern. The IR irradiation of corn starch at 20% moisture and 550 W for 90 min showed a similar change in the diffraction to V-type but with an increase in crystallinity which was attributed to the displacement of double-helical chains to a more orderly arranged structure. As in case of | 3,068,967 | 259928858 | 0 | 16 |
cereals, IR irradiated common bean showed changes in diffraction from native C-type to V type and an increase in crystallinity, which was related to the formation of amyloselipid complex. The above observations suggested that the IR treatment converts starch ultimately to V-type, irrespective of the plant source. Becker et al. (2016) [36] studied that there is a minimum dose for each application of food irradiation below which the intended effect will not be achieved. The dose requirements for some typical uses of food irradiation. Because irradiation causes only a slight temperature rise in the food being processed, it can kill microorganisms without thawing frozen food. Moreover, an effective radiation dose can be delivered through most standard food packaging materials, including those that cannot withstand heat. This means that irradiation can be applied to hermetically sealed products without the risk of recontamination or re-infestation of properly packaged foods. Some food products may have to be irradiated under special conditions, for example at low temperature or in an oxygen-free atmosphere. Others, as noted previously, may undergo multiple processing, using, for example, both ionizing radiation and heat. This particular combination may allow the use of lower radiation doses because heat makes microorganisms more sensitive to the effects of radiation. Since radiation does not damage packaging materials designed to hold food during irradiation, multiple processing is facilitated and is more economical. Insect disinfection, radiation disinfestations can contribute significantly to improving trade in certain tropical and subtropical fruits, such as citrus fruit, mangoes, and papayas. Because it affords a residue-free means | 3,068,968 | 259928858 | 0 | 16 |
of preventing the importation of harmful insects, radiation treatment offers a viable alternative to fumigation to satisfy the quarantine regulations in a number of countries. Fruit flies, for example, and even the weevil that lodges deep inside the seed of the mango can be controlled by irradiation. Shelf-life extension of perishable foods, The shelf-life of many fruits and vegetables, meat, poultry, and fish and other seafood's can be considerably prolonged -certainly doubledby treatment with combinations of refrigeration and relatively low doses of radiation that do not alter flavour or texture. Application of food irradiation Most food spoilage microorganisms are killed at doses of less than 5 kGy. Various fresh fruits, including strawberries, mangoes, and papayas, have been irradiated and marketed successfully. A combination of mild heat treatment (immersion in hot water), low-dose irradiation, and proper packaging may be successfully applied to fruits that are sensitive to higher radiation doses. Destruction of parasites, Irradiation inactivates certain parasitic organisms that are responsible for both human and animal diseases. The parasitic roundworm Trichinella spiralis, which causes trichinosis and is found in pork, is inactivated by radiation at a minimum dose of 0.15 kGy. Other parasites, including pork and beef tapeworms, the protozoon in pork responsible for toxoplasmosis, and various flukes that infest fish are rendered non-infective by low-dose radiation treatment. Conclusion Each food ingredient irradiated with a different purpose has an extra dose depending on the food its elf's characteristics. The rays commonly used to preserve food products with irradiation are gamma rays, which has its advantages and disadvantages. | 3,068,969 | 259928858 | 0 | 16 |
Chemical, nutritional, microbiological, and toxicological aspects are used for food irradiation safety parameters. The principle of radiation is excitation, ionization, and changes in components contained in foods when the radiation source touches the material. one of which is that the radiation process does not use heat so that food does not change its characteristics. However, the irradiation technique is predominately used to enhance the shelf life by inactivation of microbial activity and there is no adverse effect on nutritional value, rheological properties and functional properties of irradiated food. Thus irradiation has no adverse effect on nutritional properties of wheat flour i.e protein, fat, ash, antioxidant, phenolic content. | 3,068,970 | 259928858 | 0 | 16 |
Above Ground Drip Application Practices Alter Water Productivity of Malbec Grapevines under Sustained Deficit An objective of this study was to identify above ground drip application practices for winegrape that increase water productivity and mitigate water deficit-associated loss of yield. The influence of irrigation frequency on water productivity under two severities of sustained deficit irrigation was evaluated in field grown Malbec grapevines (Vitis vinifera L.) over three growing seasons. A weekly amount of water was delivered in a single irrigation event (1X) or apportioned into thirds and delivered in three irrigation events per week (3X). The least severe deficit (STD) had a 3-yr average maximum duty cycle (ratio of irrigation duration to irrigation interval) of 0.3, and vines irrigated 3X relative to 1X had higher water productivity each year due to a decrease in pruning weight. The most severe deficit (STD50) had a 3-yr average maximum duty cycle of 0.02 and vines irrigated 1X relative to 3X had higher water productivity due to a reduction in pruning weight in 2012 and an increase in yield in 2013. The fruit produced from vines irrigated at the frequency with highest water productivity under each deficit severity contained a lower concentration of anthocyanins. While treatment combinations did not alleviate a water-stress associated reduction in yield, results provided new information about grapevine water use efficiency that can be used to select combinations of irrigation frequencies and duty cycles with potential for increasing water productivity. Introduction A majority of global winegrape (Vitis vinifera L.) production is located in semi-arid regions where | 3,068,971 | 73571284 | 0 | 16 |
sustainability relies upon efficient use of limited water resources (Medrano et al., 2015).In wine-producing regions that experience summer drought, irrigation is commonly used to optimize vine balance and manage fruit quality (Chaves et al., 2010).However, irrigation for crop production will increasingly take place under water scarcity due to climate change and competition for water with industrial and domestic users (Costa, Ortuño, & Chaves, 2007;Fereres & Soriano, 2007;Davies, Zhang, Yang, & Dodd, 2011).Deficit irrigation is the practice of supplying an amount of water to a crop that is a fraction of the crop's estimated water demand (ET c ). Deficit irrigation is widely used as a method to sustain crop productivity under water scarcity.Sustained (SDI) and regulated (RDI) deficit irrigation and partial root-zone drying (PRD) are deficit irrigation strategies used in winegrape production to improve water use efficiency (Fereres & Soriano 2007;Medrano et al., 2015).Water productivity is the amount of marketable product produced per unit of supplied water and it is a measure of water use efficiency at the crop level (Fereres & Soriano, 2007;Davies et al., 2011;Medrano et al., 2015).The marketable product produced from winegrapes is the harvested fruit so seasonal water productivity in winegrape can be measured as the relationship between the Ravaz index (ratio of yield to pruning weight) and the amount of supplied water (Davies et al., 2011;Shellie, 2014).RDI (Shellie, 2014;Munitz, Netzer, & Schwartz, 2017).A similar level of water productivity can be attained using SDI or RDI; however, reduced water usage is usually associated with a reduction in yield (Davies et al., 2011;Shellie, | 3,068,972 | 73571284 | 0 | 16 |
2014).The PRD irrigation strategy evolved from studies with potted grapevines where it was observed that water use efficiency could be increased without reducing berry size by alternating the wet and dry portions of the root zone to induce root-to-shoot signaling of abscisic acid (ABA) (Stoll, Loveys, & Dry, 2000).A consistent response to PRD can be induced under controlled conditions; however, under field conditions, vine response to PRD has frequently been undistinguishable from that of other deficit irrigation strategies and response has been found to vary according to the supplied percentage of ET c (Dodd, 2009;Davies et al., 2011;Sadras, 2009;Chaves et al., 2010;Puértolas, Alcobendas, Alarcón, & Dodd, 2013;Romero et al., 2015). When above ground drip is used to supply water for irrigation, the drip emitters create a heterogeneous wetting pattern in the vine row beneath the drip line (Goldberg, Rinot, & Karu, 1971;Davenport, Stevens, & Whitley, 2008;Bowen, Bogdanoff, Usher, Estergarrd, & Watson, 2011).The configuration of the drip system, interval between irrigation events and irrigation amount will alter the spatial and temporal distribution of the wetting pattern in the soil profile (Sinai, Zur, & Haramati, 2007).The ideal irrigation application should recharge the soil profile in minimum time at a maximal application rate that does not cause water logging or runoff at the soil surface.The different drip irrigation application practices used under field conditions in deficit irrigation trials may partially explain the inconsistent results on vine drought response found in the literature (Sadras, 2009).In the few studies that have investigated the influence of irrigation intervals on water productivity under | 3,068,973 | 73571284 | 0 | 16 |
field conditions, results have been confounded by differing amounts of plant available water in the root zone due to different deficit irrigation treatment amounts (Hepner, Bravdo, Loinger, Cohen, & Tabacman, 1985) or irrigation frequencies with duty cycles, defined as the ratio of irrigation duration to irrigation interval, that caused excessive drainage or aeration stress from water logging at the soil surface (Goldberg et al., 1971;Selles et al., 2004).The objective of this study was to delineate the influences of above ground drip emitter configuration and irrigation event frequency on water productivity, yield components, and berry maturity in field grown Malbec grapevines under two severities of SDI.A practical goal of the study was to identify water-use-efficient, above ground drip application practices that increase water productivity under deficit irrigation by mitigating water deficit-associated loss of yield. Trial Site and Experimental Design The study was conducted in an experimental vineyard located at the University of Idaho Parma Research and Extension Center in Parma, ID (lat.43°377.9716″N,long.116°1254.1″W,750 m asl.) during the 2011, 2012 and 2013 growing seasons.The climate at this location was a Köeppen classification of BSk, meaning that plant growth was limited by water availability.Soil texture at the trial site was a sandy loam with an available water-holding capacity of 0.14 cm/cm soil.Water for irrigation was sourced from a ground well located at the trial site.The wine grape cultivar Malbec was planted in 2007 on its own roots in rows oriented north to south with a row and vine spacing of 2.4 m and 1.8 m, respectively.The vines were double-trunked and | 3,068,974 | 73571284 | 0 | 16 |
each trunk was trained to form one side of a bilateral cordon.Canes were spur-pruned annually to 16 buds/m of cordon.Shoots were vertically positioned on a two-wire trellis with moveable wind wires.Disease, weed and pest control were managed according to local commercial practices. The experimental design was a 2 × 3 factorial, split-split plot with six replicate blocks.Irrigation amount was the main factor.Drip line emitter spacing/delivery rate was split within each irrigation amount.Irrigation event frequency was split within each drip line configuration subplot.A block was comprised of 12 adjacent vine rows with 12 vines per row.Six consecutive vines across the 12 adjacent rows of each block were deficit irrigated from fruitset until harvest with 70 (STD) or 35% (STD50) of estimated weekly ET c .The STD irrigation amount was intended to induce a sustained, mild water deficit, similar to standard industry practice (Keller, Smithyman, & Mills, 2008).Weekly ET c was calculated by multiplying the value for reference crop evapotranspiration (ET r ), acquired from a weather station located within 3 km of the study site (http://www.usbr.gov/pn/agrimet/wxdata.html),by the value of a crop coefficient that was increased from 0.3 to 0.7 during the growing season (Allen, Pereira, Raes, & Smith, 1998;Keller et al., 2008).Different spacing between in-line emitters with different delivery rates were used to create spatially different soil wetting patterns.Six adjacent rows within each irrigation subplot had above ground drip with an emitter spacing/delivery rate of 45 cm/16.7 ml/min or 90 cm/33.4ml/min.Both emitter spacing/delivery rate configurations delivered the same amount of water per hour.Plots with 45 cm emitter | 3,068,975 | 73571284 | 0 | 16 |
spacing had four emitters per vine (4E) and plots with 90 cm emitter spacing had two emitters per vine (2E).Each drip line configuration was used to supply a weekly amount of water as a single weekly event (1X) or apportioned into thirds and delivered as three irrigation events per week (3X).Three adjacent rows (6 vines per row) with the same drip emitter configuration were irrigated 1X or 3X.Vines located in outer rows and at either end of the interior row of each plot were treated as guard vines.Data were collected from vines located in the interior of the middle row of each plot.The trial perimeter had a two-vine deep border.Allocation of irrigation amount, emitter spacing/delivery configuration and irrigation event frequency treatment levels was randomized within each replicate block and the same treatment level was applied to all plots in each successive year of the study.The six replicate blocks for each treatment level were connected to one of eight, independently controlled, water supply manifolds.The manifolds were equipped with a programmable solenoid and a flow meter.All plots were irrigated to field capacity prior to budbreak.There were no subsequent irrigations until the start of treatment applications.The irrigation treatments were initiated after fruitset, when berries were ~7 mm in diameter and vines were at growth stage 31 of the modified E-L grapevine growth stage system (Coombe, 1995). Soil Moisture and Vine Water Status Soil moisture was recorded hourly using a time domain reflectometry (TDR) data-logging system (Moisture Point; Environmental Sensors Inc. Sydney, Canada).Each probe was 91-cm long and had measurement | 3,068,976 | 73571284 | 0 | 16 |
sensors located at soil depths of 30, 46, 61, 76, and 91 cm.One probe was permanently installed at a standard distance from the vine trunk and drip emitter, as described by Bowen, Bogdanoff, and Estergaard (2012), in a single replicate of each subplot. Vine water status was monitored weekly by measuring leaf water potential at midday (Ψ lmd ) using a pressure chamber (model 610; PMS Instruments; Corvallis, OR) as described by Shellie (2014).Weekly Ψ lmd was measured on the sixth day after a weekly irrigation event.In 2013, the 13 C/ 12 C ratio (δ 13 C) of juice at harvest was measured following the method of Herrero-Langreo, Tisseyre, Goutouly, Scholasch, and Van Leeuwen (2013). Yield Components and Berry Composition Fruit were harvested when a composite sample of randomly collected clusters had a target soluble solids concentration (SS) of ~24% and a juice titratable acidity (TA) of 4 to 6 g/L.All plots were harvested on the same day.On the day of harvest, a basal cluster was removed from either side of two main shoots from the center two data vines in each plot (n = 8/plot).The sampled clusters were immediately placed into a cooler and transported to the lab on the day of harvest.The remaining clusters on each data vine were counted as they were removed from the vine and their weight was added to the weight of sampled clusters to determine yield per vine.Sampled clusters were individually weighed and used to calculate average cluster weight.Average berry fresh weight and number of berries per cluster were | 3,068,977 | 73571284 | 0 | 16 |
determined by counting the number of berries per cluster and dividing the weight of the cluster by the number of berries per cluster (2011 and 2012) or by dividing average cluster weight by the average weight of 100, randomly sampled berries (2013).Samples of 100 berries were stored at -80 °C for analysis of total berry anthocyanins following the method of Iland, Bruer, Edwards, Weeks, & Wilkes (2004), and for δ 13 C analysis by isotope-ratio mass spectrometry at the Stable Isotope Facility (UC Davis, University of California Davis, CA). The remaining berries from the 8-cluster sample were used to measure juice SS, pH and TA following methods of Iland et al. (2004) using equipment previously described by Shellie (2006).The same vines harvested for yield and berry measurements were pruned to two bud spurs during dormancy and pruned canes from each vine were weighed.The ratio of yield to pruning weight (Ravaz index) was calculated as an indicator of vine balance.Water productivity was expressed as the relationship between the Ravaz index and seasonal irrigation amount. Seasonal cumulative growing degree days (GDD) were calculated from daily maximum (no upper limit) and minimum temperatures from 1 Apr to 31 Oct using a base threshold of 10 °C.Temperature data were obtained from the same weather station used for ET r . Statistical Analysis Data were analyzed by year using a mixed model analysis of variance with a 2 × 3 factorial and split-split plot treatment structure (SAS version 8.02; SAS Institute, Cary, NC).Fixed effects were irrigation amount, emitter configuration, and irrigation | 3,068,978 | 73571284 | 0 | 16 |
frequency.Weekly Ψ lmd were analyzed by phenological stage of the vine.Probability of a significant difference among fixed effect treatment levels (p ≤ 0.05) was determined using the Tukey-Kramer adjusted t-test.The significance of interaction effects was determined using the LSMEANS slice statement at p ≤ 0.05.Graphs presented in figures were generated using Sigmaplot 11.2 (Systat Software, San Jose, CA). Environmental Conditions and Irrigation Amounts In 2011, seasonal GDD accumulation and ET r were lowest of the three study years and were 14 and 7% lower than the 11-yr average for the location (Table 1).In 2012, GDD and seasonal ET r were similar to the 11-yr site average.In 2013, seasonal GDD was similar to the 11-yr site average but ET r was 6% higher than the 11-yr site average.The amount of seasonal precipitation was similar to the 11-yr site average in each study year.The onset of bloom and the day of harvest in 2011 were 20 and 27 days later than in 2013.In 2011, 2012 and 2013 the day of year for bloom was 175, 167, and 155; for veraison was 241, 233 and 224; and for harvest was 293, 284 and 266, respectively.The elapsed number of days between bloom and veraison was 66 (2011 and 2012) and 69 (2013); and between veraison and harvest was 51 (2011 and 2012) and 42 (2013).Irrigation treatments were initiated on day of year 173, 179 and 184 and were applied for 15, 14 and 12 weeks in 2011, 2012 and 2013, respectively.The amount of water supplied to vines under the STD | 3,068,979 | 73571284 | 0 | 16 |
irrigation amount was 37, 45 and 35% of seasonal ET r in 2011, 2012 and 2013, respectively (Table 1).Vines under the STD50 irrigation amount were supplied 50 (2011), 43 (2012) and 50% (2013) of the STD irrigation amount.The amount of water actually delivered to 3X plots was from 2 to 7% less than 1X plots.The amount of water delivered to plots with 4E drip line configuration was ~1 to 5% less than plots with 2E drip line configuration.Note. a Average and standard deviation for years 2000 through 2010.b Heat unit accumulation (GDD), precipitation (Pcp) and reference evapotranspiration (ET r ) from the Bureau of Reclamation AgriMet system [(www.usbr.gov/pn/grimet/),latitude 43°4800, longitude 116°5600″, elevation 700 m] PMAI weather station.GDD calculated as simple average with no upper limit and a base threshold of 10 °C.ET r from 1982 Kimberly-Penman equation for well-watered alfalfa with 30-50 cm top growth (Jensen et al., 1990).c Amount of water supplied to Malbec grapevines to satisfy 70 (STD) or 35 (STD50) percent of estimated water demand. Weekly estimated irrigation amount was delivered in a single weekly event (1X) or apportioned into thirds and delivered as three events per week (3X) using above ground drip with in-line emitter spacing/delivery rate configurations of 45 cm/16.7 ml/min (4E) or 90 cm/33.4ml/min (2E). Soil Moisture Irrigation amount, drip line configuration and irrigation event frequency influenced the vertical depth of water penetration after an irrigation event.Water penetration was deeper under STD (72 to 91 cm) than under STD50 (46 to 61 cm) and deeper in plots with 2E than | 3,068,980 | 73571284 | 0 | 16 |
4E drip configuration.The average depth of water penetration in plots under STD with 2E drip configuration was 91 cm when irrigated 1X and 76 cm when irrigated 3X.Plots under STD50 with 2E drip configuration had an average depth of water penetration of 61 cm when irrigated 1X and 46 cm when irrigated 3X.In plots with 4E drip configuration, the average depth of water penetration was 76 cm under STD and 46 cm under STD50, and was similar when irrigated 1X or 3X. Leaf Water Potential and δ 13C Weekly Ψ lmd was measured eight (2011 and 2012) or six (2013) times between fruitset and veraison and five times (all years) between veraison and harvest.The influence of irrigation event frequency on Ψ lmd was different under each irrigation treatment amount (Figure 1).Under STD, vines irrigated 3X had significantly less negative Ψ lmd than vines irrigated 1X, during the period from fruitset to veraison in 2011 and 2012, and during the period from veraison to harvest in 2012 and 2013.In all three years, the Ψ lmd of vines under STD50 irrigated 3X was similar, during the period from fruitset to veraison, and significantly different, during the period from veraison to harvest, as vines irrigated 1X.In 2013, irrigation frequency had no influence on δ 13 C; however, vines under STD50 had a lower δ 13 C (-24.13) than vines under STD irrigation (-26.65).Emitter configuration had no significant influence on Ψ lmd . Figure 1.Weekly midday leaf water potential of Malbec grapevines under sustained deficit irrigation that were supplied 70 | 3,068,981 | 73571284 | 0 | 16 |
(STD) or 35 (STD50)% of estimated vine water demand as a single weekly event (1X) or apportioned into thirds and applied as three events per week (3X) and delivered with above drip that had an emitter spacing/delivery rate of 45 cm/16.7 ml/min (4E) or 90 cm/33.Midday leaf water potential (MPa) Vine Balance and Water Productivity There was a significant interaction between irrigation frequency and irrigation amount on pruning weight each year and on yield in one out of two years (Figure 2).In each respective year, vines under STD irrigated 3X had 19, 11, and 16% lower pruning weight than vines irrigated 1X.The only year that irrigation frequency had a significant influence on yield under STD was in 2012, when vines under STD irrigated 3X had 21% higher yield than vines irrigated 1X (Figure 2).In 2012, the berry fresh weight of vines under STD irrigated 3X was greater than that of vines irrigated 1X (Table 2).Vines under STD irrigated 3X had greater cluster weight in 2011 and 2013 and lower berry weight in 2011 than vines irrigated 1X (Table 2); however, these differences had no detectable influence on yield in 2011 or 2013 (Figure 2).Under STD50, irrigation frequency influenced pruning weight only in 2012 when vines irrigated 1X had 24% lower pruning weight than vines irrigated 3X (Figure 2).In 2013, vines under STD50 irrigated 1X had higher yield than vines irrigated 3X.In 2013, vines under STD50 irrigated 1X had greater cluster weight and a greater number of berries per cluster than vines irrigated 3X (Table 2). The | 3,068,982 | 73571284 | 0 | 16 |
dormant pruning weight of vines under STD50 was 34 (2011) or 50% (2012 and 2013) less than that of vines under STD (Figure 2).Vines under STD50 had 43 and 31% lower yield than vines under STD in 2012 and 2013, respectively, and similar (5.5 kg) yield per vine in 2011 (Figure 2).In 2012 and 2013, vines under STD50 had ~26% fewer clusters than vines under STD (Table 2).Vines under STD50 had lower cluster weight and berry fresh weight than vines under STD in each respective year of the study; although the difference in cluster weight in 2013 was not of statistical significance.Drip line configuration had no consistent main or interactive effect on pruning weight or yield (data not shown) or yield components (Table 2).Table 2. Yield components for Malbec grapevines supplied with 70 (STD) or 35 (STD50) percent of estimated weekly water demand in a weekly event (1X) or apportioned into three events per week (3X) using above ground drip tubing with emitter spacing/delivery rate configurations of 90 cm/33.4ml/min.(2E) or 45 cm/16.7 ml/min (4E) over three growing seasons in Parma, ID Figure 2. Relationship between pruning weight and yield in Malbec grapevines under sustained deficit irrigation that supplied 70 (STD) or 35 (STD50)% of estimated vine water demand as a single weekly event (1X) or apportioned into thirds and applied as three events per week (3X).Error bars represent the standard error of the mean Irrigation frequency had a significant influence on the Ravaz index and the effect differed by irrigation amount (Figure 3).More frequent irrigation significantly | 3,068,983 | 73571284 | 0 | 16 |
increased the Ravaz index of vines under STD in all years, but decreased the Ravaz index of vines under STD50 in two out of three years.Vines under STD irrigated 3X had a higher Ravaz index than that of vines irrigated 1X by 37, 31 and 26% in each respective year.Vines under STD50 irrigated 3X had a lower Ravaz index than vines irrigated 1X by 28 and 23% in 2012 and 2013, respectively.Irrigation amount had no significant effect on the Ravaz index in 2011 or 2012.In 2013 the Ravaz index was significantly higher in vines under STD50 (4.6) than in vines under STD (3.4).In 2011 and 2012, the Ravaz index was 3.9 and 3.2, respectively.Drip line configuration had no consistent influence on the Ravaz index. Pruning weight per vine (kg) Berry Composition The SS and pH of juice at harvest were similar among all treatments (data not shown).In each respective year, SS was 23, 25, and 22% at the time of harvest; and pH was 4.0, 3.9, and 3.7.Irrigation event frequency and drip emitter configuration had no consistent main or interactive effects on juice TA; however, the TA of juice from vines under STD50 was lower each year than vines under STD (Table 3). The influence of irrigation event frequency on the concentration of anthocyanins and phenolics was inconsistent among years and differed by irrigation amount (Table 3).In vines under STD, the concentration of anthocyanins in berries irrigated 3X was lower in 2011 and 2012 than that of vines irrigated 1X.The concentration of phenolics in vines under | 3,068,984 | 73571284 | 0 | 16 |
STD irrigated 3X was similar in 2011 and 2013 and lower in 2012 than vines irrigated 1X.Under STD50, the concentration of anthocyanins and phenolics in berries from vines irrigated 3x was higher than that of vines irrigated 1X in each year of the study, though the difference in anthocyanins was not of statistical significance in 2012.Berries from vines under STD50 had a higher concentration of anthocyanins and phenolics in 2012 and 2013 than that of vines under STD (Table 3). Discussion A practical goal of this study was to identify water use efficient, above ground drip application practices that mitigate undesirable, water deficit-associated loss of yield in winegrape.The irrigation event intervals evaluated in this study under each severity of deficit irrigation alleviated water stress-associated yield reduction in only one out of three years; however, the observed interaction between irrigation amount and event frequency on water productivity provides new information about irrigation frequency and duty cycle combinations that influence water productivity under SDI. In this study, irrigation amount and event frequency influenced the vertical depth of moisture in the soil profile. The distribution of soil moisture under drip irrigation has been described as mainly two dimensional, with soil moisture content highest beneath the drip line and decreasing laterally (Goldberg et al., 1971;Selles et al., 2004;Davenport et al., 2008;Bowen et al., 2012).A model using irrigation frequency and irrigation duty cycle, defined as the ratio of irrigation duration to irrigation interval, was used by Sinai et al. (2007) to predict maximum depth of water penetration under drip irrigation.Depth of | 3,068,985 | 73571284 | 0 | 16 |
water penetration was shown to increase with less frequent irrigation or with increasing duty cycle values (Sinai et al., 2007).In this study, plots irrigated 1X and 3X under each irrigation amount had similar duty cycle values.The 3-yr average maximum duty cycle value under STD was 0.30 and under STD50 was 0.02.The different duty cycle values for STD and STD50 and different irrigation frequency in 1X and 3X plots explain the greater depth of water penetration observed under STD relative to STD50 and in plots irrigated 1X relative to 3X. The author found three published field studies with winegrapes in which above ground drip irrigation interval was evaluated as a treatment (Goldberg et al., 1971;Hepner et al., 1985;Selles et al., 2004).In the study of Hepner et al. (1985), the main effect of irrigation frequency could not be evaluated because it was confounded within different severities of SDI and RDI.In Goldberg et al. (1971) and Selles et al. (2004), an amount of water equal to estimated demand (well-watered) was delivered at irrigation frequencies similar to the 1X and 3X frequencies evaluated in this study.In both of these studies, depth of water penetration increased with less frequent irrigation.Goldberg et al. (1971) and Selles et al. (2004) found that irrigation frequency influenced water productivity and that higher productivity was due to an increase in yield that was proportionally greater than the increase in pruning weight.Goldberg et al. (1971) found that water productivity increased as irrigation frequency increased, whereas Selles et al. (2004) reported an inverse relationship between water productivity and | 3,068,986 | 73571284 | 0 | 16 |
irrigation frequency.The contrasting results of these two studies could be due to different soil textures and/or the amount of supplied water relative to actual vine water demand. The influence of irrigation frequency on water productivity in this study differed according to deficit irrigation amount.An interaction between irrigation amount and application timing on drought response has also been noted by Romero et al. (2015).Under the least severe water deficit (STD), more frequent irrigation increased water productivity each year and the increase was due to a decrease in pruning weight.An explanation for the reduction in pruning weight of vines under STD irrigated 3X is not readily apparent from Ψ lmd values.Reduction of shoot growth is usually associated with a Ψ lmd value ≤ -1.0 MPa (Shellie, 2006).However, in this study, vines under STD irrigated 3X had values of Ψ lmd more negative than -1.0 MPa only in 2013.Under STD, the fruit produced from plots with highest water productivity had a lower concentration of anthocyanins in 2011 and 2012. The lower concentration of anthocyanins could be attributed to the higher values of Ψ lmd between fruitset and veraison in 3X relative to 1X irrigated plots. Under the most severe water deficit (STD50), less frequent irrigation was associated with increased water productivity in 2012 and 2013.The increase in 2012 was due to a decrease in pruning weight and the increase in 2013 was due to an increase in yield.The decrease in pruning weight in 2012 was most likely due to the more negative Ψ lmd of vines irrigated 1X relative | 3,068,987 | 73571284 | 0 | 16 |
to 3X.The greater number of berries per cluster observed in 1X plots under STD50 in 2013 might have been related to the less negative Ψ lmd in the first weeks after fruitset which could have reduced the amount of water stress-associated berry abscission (May, 2004).Under STD50, the irrigation frequency with highest water productivity had the lowest concentrations of anthocyanins and phenolics.This lower concentration was not due to a lack of vine water stress, as vines irrigated 1X had lower Ψ lmd than vines irrigated 3X.The concentration of phenolics may have been lower in vines under STD50 irrigated 1x relative to 3X because of more frequent exposure to high temperatures.An increase in canopy temperature under deficit irrigation has been associated with lower anthocyanin accumulation (Shellie & King, 2013).The cumulative duration of exposure to damaging high temperatures may have been greater under 1X than 3X due to the longer interval between irrigation events. Under each irrigation amount, increased water productivity was accompanied by an increase in yield in only one out of three years.An increase in yield and decrease in pruning weight occurred only under STD in 2012 in plots irrigated 3X.This was also the only year when a significant difference in Ψ lmd was maintained throughout berry development between vines irrigated 1X and 3X.The increase in berry fresh weight in 2012 in vines under STD irrigated 3X was likely attributed to higher vine water status.The general lack of yield response to the irrigation frequencies and duty cycles evaluated in this study suggests that the pattern of soil | 3,068,988 | 73571284 | 0 | 16 |
moisture distribution associated with the treatment combinations evaluated in this study did not induce the same drought response observed in potted vines under partial root-zone drying (Stoll et al., 2000;Sadras, 2009). The influence of deficit irrigation severity on yield components, berry maturity and berry composition observed in this study were similar to that reported by others (Shellie, 2006;Shellie, 2014;Romero et al., 2015;Munitz et al., 2017).The irrigation intervals evaluated in this study did not alleviate a water-stress associated reduction in yield under either severity of deficit irrigation; however, study results provide new information about water use efficiency that can be used to identify combinations of irrigation frequencies and duty cycles with potential for increasing water productivity. Figure1.Weekly midday leaf water potential of Malbec grapevines under sustained deficit irrigation that were supplied 70 (STD) or 35 (STD50)% of estimated vine water demand as a single weekly event (1X) or apportioned into thirds and applied as three events per week (3X) and delivered with above drip that had an emitter spacing/delivery rate of 45 cm/16.7 ml/min (4E) or 90 cm/33.4ml/min (2E) in 2011(A and B), 2012 (C and D), and 2013 (E and F).The day of veraison is indicated with the letter "V" Note. a Different lower case letters indicate significant difference between treatment levels within a given year (p ≤ 0.05 determined by Tukey-Kramer adjusted t-test).b Least square mean values followed by a different letter between subplot treatment level rows within a year column are significantly different (p ≤ 0.05) according to LSMEANS slice statement using a mixed | 3,068,989 | 73571284 | 0 | 16 |
model analysis of variance.c *, p ≤ 0.05; **, p ≤ 0.01; ns, not significant. Table 1 . Growing season (1 Apr through 31 Oct) environmental conditions at the field trial site and irrigation treatment amounts Note. a Different lower case letters indicate significant difference between main effect treatment levels within a given year (p ≤ 0.05 determined by Tukey-Kramer adjusted t-test).b Least square mean values followed by a different letter between subplot treatment level rows within a year column are significantly different (p ≤ 0.05) according to LSMEANS slice statement in mixed model analysis of variance.c *, p ≤ 0.05; **, p ≤ 0.01; ns, not significant. | 3,068,990 | 73571284 | 0 | 16 |
Carbohydrate metabolism and fertility related genes high expression levels promote heterosis in autotetraploid rice harboring double neutral genes Background Autotetraploid rice hybrids have great potential to increase the production, but hybrid sterility is a major hindrance in the utilization of hybrid vigor in polyploid rice, which is mainly caused by pollen abortion. Our previous study showed that double pollen fertility neutral genes, Sa-n and Sb-n, can overcome hybrid sterility in autotetraploid rice. Here, we used an autotetraploid rice line harboring double neutral genes to develop hybrids by crossing with auto- and neo-tetraploid rice, and evaluated heterosis and its underlying molecular mechanism. Results All autotetraploid rice hybrids, which harbored double pollen fertility neutral genes, Sa-n and Sb-n, displayed high seed setting and significant positive heterosis for yield and yield-related traits. Cytological observations revealed normal chromosome behaviors and higher frequency of bivalents in the hybrid than parents during meiosis. Transcriptome analysis revealed significantly higher expressions of important saccharides metabolism and starch synthase related genes, such as OsBEIIb and OsSSIIIa, in the grains of hybrid than parents. Furthermore, many meiosis-related and specific genes, including DPW and CYP703A3, displayed up-regulation in the hybrid compared to a parent with low seed setting. Many non-additive genes were detected in the hybrid, and GO term of carbohydrate metabolic process was significantly enriched in all the transcriptome tissues except flag leaf (three days after flowering). Moreover, many differentially expressed genes (DEGs) were identified in the yield-related quantitative trait loci (QTLs) regions as possible candidate genes. Conclusion Our results revealed that increase in the number | 3,068,991 | 149445636 | 0 | 16 |
of bivalents improved the seed setting of hybrid harboring double pollen fertility neutral genes. Many important genes, including meiosis-related and meiosis-specific genes and saccharides metabolism and starch synthase related genes, exhibited heterosis specific expression patterns in polyploid rice during different development stages. The functional analysis of important genes will provide valuable information for molecular mechanisms of heterosis in polyploid rice. Electronic supplementary material The online version of this article (10.1186/s12284-019-0294-x) contains supplementary material, which is available to authorized users. Background Heterosis, or hybrid vigor, is a complex biological phenomenon, which is improved or superior phenotypic performance of hybrid in comparison to one or both parents, such as enhanced grain yield, stress tolerance and biomass production. Heterosis has been extensively applied to increase the rice yield in the world (Cheng et al. 2007). However, productivity of rice has been stagnant in the past few years. Polyploid species play an important role in breeding programs, such as cotton (Flagel et al. 2008), wheat (Goncharov et al. 2007), and rapeseed (Albertin et al. 2006). Rice polyploidization is an effective method to increase the size of rice genome and improve the wide adaptability (Cai et al. 2001;Guo et al. 2017). The polyploid rice hybrids showed stronger biological advantage and yield potential compared with diploid rice hybrids, and has attracted the attention of many rice researchers (Shahid et al. 2011;Wu et al. 2013;Guo et al. 2017). However, autotetraploid rice has many unfavorable traits, especially low seed setting, which limits its commercial utilization (Shahid et al. 2010(Shahid et al. , 2013aChen et | 3,068,992 | 149445636 | 0 | 16 |
al. 2018a). Recently, our research team found that polyploidy enhanced pollen sterility loci interactions and increased chromosomal abnormalities in autotetraploid hybrid rice (Wu et al. 2015), and also revealed that intersubspecific diploid and autotetraploid hybrid rice sterility could be overcome by double neutral genes (Shahid et al. 2013b;Wu et al. 2017). After years of efforts, our research group have developed few neo-tetraploid rice lines with high seed setting (> 80%) (Guo et al. 2016(Guo et al. , 2017, and two new neo-tetraploid rice have been registered for the PVP (Protection for new varieties of plant) in China . Moreover, neo-tetraploid rice could overcome the sterility and produce high heterosis in autotetraploid hybrid rice (Guo et al. 2017). Two photoperiod-and thermo-sensitive genic male sterile lines (PS006 and PS012) of polyploid rice showed stronger hybrid vigor and great potential for improving rice quality and productivity ). Next generation high-throughput sequencing, such as RNA sequencing (RNA-seq) and microarray technology, is widely used to investigate gene expression and function. The RNA-seq enabled us to understand differentially expressed genes associated with abiotic stresses and pollen development in diploid rice (Jin et al. 2013;Hu et al. 2016;Fu et al. 2017). The RNA-seq has also been applied to detect differentially expressed genes between diploid and autotetraploid rice during pollen development Chen et al. 2018a;Li et al. 2018). Furthermore, RNA-seq has been widely used to investigate heterosis in various plants, such as wheat , maize (Ma et al. 2018), rapeseed (Shen et al. 2017), tobacco (Tian et al. 2018) and rice (Wei et al. | 3,068,993 | 149445636 | 0 | 16 |
2009). The differentially expressed genes were found to be closely associated with heterosis in super rice LYP9 and its parents through RNA-seq (Wei et al. 2009). Later, many differentially expressed genes closely related to root heterosis at tillering and heading stage were detected in super hybrid XY9308 and its parents through RNA-seq (Zhai et al. 2013). Chen et al. (2018b) compared the transcriptomes between super hybrid Wufengyou T025 (WFYT025) and its parents during young panicle development, and suggested that carotenoid biosynthesis and plant hormone signal transduction were enriched in differentially expressed genes, and these genes were related to the grain number heterosis. A number of genes associated with leaf, anthers and ovary heterosis in neo-tetraploid rice hybrids were identified by RNA-seq, which were related to photosynthesis and metabolic process and transport (Guo et al. 2017). Our previous study indicated that saccharide abnormal distribution and down-regulation of saccharide transport genes may cause pollen sterility and lead to low seed setting in autotetraploid rice (Chen et al. 2018a). In this study, to increase yield of autotetraploid rice, we used an autotetraploid rice line harboring double neutral genes for pollen fertility at Sa and Sb loci, which could overcome F 1 sterility when it crossed with low fertility autotetraploid rice Chen et al. 2018a). We thus primarily aimed to evaluate heterosis mechanism of neo-tetraploid and autotetraploid rice harboring double neutral genes, and to observe the role of chromosome configuration and behavior in heterosis and fertility. In addition, we detected differentially expressed genes between parents and hybrid in nine tissues | 3,068,994 | 149445636 | 0 | 16 |
at three development stages using RNA-seq, which would provide insights into the molecular mechanism underlying heterosis in autotetraploid and neo-tetraploid rice, and provide new germplasm for polyploid rice breeding. Results Heterosis evaluation of hybrids generated by crossing of autotetraploid with neo-tetraploid rice Analysis of the agronomic traits of five hybrids, which were developed by crossing autotetraploid rice line (T449) with five neo-tetraploid rice lines, showed significant improvement in important yield-related traits, including number of filled grains per plant, yield per plant and seed setting. Evaluation of heterosis indicated that the values for mid-parent heterosis (MPH) were positive for all the traits except grain length and total grains per plant, and the highest MPH was found for grain yield per plant (170.89%). The high-parent heterosis (HPH) values were positive for the filled grains and grain yield per plant, and the highest HPH was detected for filled grains per plant (71.10%) (Additional file 1: Table S1). Then, we further selected a hybrid (T449 × H1) for transcriptome analysis to analyze the heterosis mechanism in detail. The hybrid had high seed setting, pollen and embryo sac fertility ( Fig. 1; Table 1 and Additional file 2: Table S2), although the maternal line, T449, had low pollen fertility and seed setting. The hybrid displayed significant positive MPH for all the traits, and the values for MPH were very high for filled grains per plant, grain yield per plant and seed setting (Table 1). Meanwhile, the hybrid also showed positive HPH values for most of the traits except effective number of | 3,068,995 | 149445636 | 0 | 16 |
panicles per plant, 1000-grain weight, and grain length and width (Table 1). Chromosome configuration at diakinesis and metaphase I in F 1 hybrid compared to its parents The tetravalent was the most type of chromosome configuration at diakinesis and metaphase I in the parents. The numbers of bivalent chromosomes were higher in H1 (high seed setting) than T449 (low seed setting), and univalent type of chromosomes were higher in T449 than both H1 and F 1 (Table 2, Fig. 2). However, bivalent chromosomes were the most frequent in F 1 (Table 2), and 9.07 and 12.57 bivalents were found in each pollen mother cell (PMC) at diakinesis and metaphase I, respectively. There were significant differences in the numbers of chromosome configurations between F 1 hybrid and parents, and these results indicated that the increase of bivalent type may improve pollen fertility and seed setting. The chromosome configurations of tetravalent types were divided into five types, including ring shape (Fig. 3a1-a4), chain shape ( Fig. 3b1-b4), frying Fig. 1 Comparisons of morphological characteristics between F 1 hybrid and its parents. a Plant appearance of F 1 , T449 and H1. Pollens of T449 (b), F 1 (c), and H1 (d), blue arrows indicate normal pollens, red arrows indicate abnormal pollens. Bar = 100 μm. Embryo sacs of T449 (e), F 1 (f) and H1 (g), white arrows indicate antipodal cells and red arrows indicate two polar nuclei. Bar = 100 μm pan shape ( Fig. 3c1-c4), "X" shape ( Fig. 3d1-d4) and "OK" shape ( Fig. 3e1-e4). | 3,068,996 | 149445636 | 0 | 16 |
The frequency of ring shape was the most frequent chromosome configuration of tetravalent types at diakinesis and metaphase I (Table 2). Differentially expressed genes in F 1 hybrid and parents In order to investigate transcriptome changes in F 1 and its parents, the transcriptome profiles of F 1 and parents were analyzed in nine tissues, including anthers (P1) and flag leaves (L1) at meiosis stage, and flag leaves (L2), leaf sheath (Z2), anther (P2) and embryo sac (E2) at pre-flowering stage, and flag leaves (L3), leaf sheath (Z3) and grain (P3) at three days after flowering (Additional file 4: Figure S2). In total, more than 3.7 billion clean reads were detected in different samples. We aligned clean reads against the Nipponbare reference genome (MSU 7.0), and 92.52% to 96.32% annotated transcripts of the reference genome was obtained in our materials (Additional file 6: Table S4). The correlation for the gene expression level from three biological replicates of each line was more than 0.8 (Additional file 7: Table S5), and principal component analysis (PCA) indicated that three biological replicates were clustered together, and flag leaves and leaf sheath were also clustered together (Additional file 8: Figure S3). The correlations between F 1 and its parents were investigated in different samples by hierarchical cluster analysis using Cluster 3.0 software. The results demonstrated that F 1 and its parents always assembled into primary groups at the same tissue, and the transcriptome profiles of F 1 were similar to H1, and these results were consist with the morphological and cytological | 3,068,997 | 149445636 | 0 | 16 |
observations (Additional file 9: Figure S4). A total of 12 DEGs were randomly selected for qRT-PCR validation. We compared the qRT-PCR results, and the expression trends were consistent with RNA-seq data, and a correlation coefficient was R 2 = 0.8806 (Additional file 10: Figure S5), which demonstrated that RNA-seq data is reliable. Differentially expressed anther specific genes were associated with meiosis stage-specific genes in F 1 compared to its parents T449 is an autotetraploid rice line with low fertility and H1 is a neo-tetraploid rice line with high fertility, so we specifically investigated meiosis-specific genes associated with high fertility. We compared expression levels of genes between F 1 vs T449 and H1 vs T449, and 381 genes were found to be commonly up-regulated between both comparisons in anthers during meiosis (Additional file 16: Table S8). GO analysis of these 381 genes showed that six biological process categories and nine molecular function categories were significantly enriched (Additional file 17: Fig. 4 Gene ontology (GO) enrichment heat map for DEG FPU in 9 tissues (GO terms were selected based on their appearance at least in three tissues or more). L1 and P1 represent flag leaves and anthers at meiosis stage, respectively. L2, P2, E2 and Z2 represent flag leaves, anther, embryo sac and leaf sheath at pre-flowering stage, respectively. L3, P3 and Z3 represent flag leaves, grain and leaf sheath at three days after flowering, respectively Table S9). We compared 381 up-regulated genes with microarray data of wild type rice anther meiosis stage-specific expression, and meiosis-related genes (Fujita | 3,068,998 | 149445636 | 0 | 16 |
et al. 2010;Tang et al. 2010;Deveshwar et al. 2011;Yant et al. 2013;Luo et al. 2014;Wright et al. 2015), and identified 4 meiosis-related genes including LOC_Os09g32020 (OsDFR), LOC_Os01g68870 (MSP1) and LOC_Os12g24420 and LOC_Os10g06770 (CDKG1), and 26 meiosis-specific expressed genes (Additional file 18: Table S10). Four pollen-related genes were identified from further analysis of 26 meiosis-specific expressed genes, including LOC_ Os03g07140 (DPW), LOC_Os04g24530 (OsACOS12), LOC_Os06g40550 (OsABCG15; PDA1) and LOC_ Os08g03682 (CYP703A3). Among 381 genes, 47 genes exhibited down-regulation in T449 compared to its diploid counterpart (E249) (Chen et al. 2018a) (Fig. 6a). Interestingly, two meiosis-related and 19 meiosis-specific genes were identified in these genes (Additional file 19: Table S11), including LOC_Os09g32020 (OsDFR) and LOC_ Os12g24420 (CDKG1), and analysis of 19 meiosis-specific genes revealed four pollen-related genes, including LOC_Os03g07140 (DPW), LOC_Os04g24530 (OsA-COS12), LOC_Os06g40550 (OsABCG15; PDA1) and LOC_Os08g03682 (CYP703A3). We performed the predicted protein-protein interactions of 47 genes using STRING v10, and the results showed that 17 genes constituted genetic sub-networks, including one meiosis-related and six meiosis-stage specific genes. The meiosis-related gene (OsDFR, LOC_Os09g32020) interacted with two meiosis-specific gene, including LOC_Os08g03682 (CYP703A3), encodes cytochrome P450 hydroxylase and LOC_Os04g24530 (OsACOS12), encodes acyl-CoA synthetase 12, which interacted with six differently expressed genes, including three cytochrome P450, two AMP-binding enzyme and a protein binding protein (Fig. 6b). Human Diseases Endocrine and metabolic diseases 3 2 3 2 1 1 1 0 1 * and ** denote significant enrichment of DEG FPU among functional categories with P < 0.05 and P < 0.01, respectively. L1 and P1 represent flag leaves and anthers at meiosis | 3,068,999 | 149445636 | 0 | 16 |
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