Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l^–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.     

Cysteine pKa depression by a protonated glutamic acid in human DJ-1

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined p K a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p K a of 5.4 +/- 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine p K a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the p K a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its p K a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.     

烟粉虱B生物型若虫、皮蜕及成虫提取物对双斑蚜小蜂行为的影响

烟粉虱B生物型的若虫、皮蜕及其成虫的提取物作为一种利它素信息源,在室内对其在双斑蚜小蜂寻找寄主取食、寄生行为的影响进行了生物测定。烟粉虱的若虫、皮蜕及其成虫分别用正己烷、乙醇和无菌水进行粗提。研究结果发现,双斑蚜小蜂在处理区寻找寄主停留的时间高于对照区。在处理区,双斑蚜小蜂行动活泼,对利它素源表现出高的正趋向性和选择性。对于同一利它素源、同一提取介质的两种不同浓度,双斑蚜小蜂在若虫 水提取物的高浓度区停留的时间（111.23s）最长,与在低浓度区的停留时间差异显著;而在烟粉虱皮蜕及其成虫的水、正己烷和乙醇提取物处理区,不同浓度的提取物对蚜小蜂停留的时间影响差异不显著。本研究的结果表明,利它素可以增加蚜小蜂寻找寄主的效率,有利于蚜小蜂寻找到适宜的寄主。     

The Influence of Diabetic Peripheral Neuropathy on Local Postural Muscle and Central Sensory Feedback Balance Control

Poor balance control and increased fall risk have been reported in people with diabetic peripheral neuropathy (DPN). Traditional body sway measures are unable to describe underlying postural control mechanism. In the current study, we used stabilogram diffusion analysis to examine the mechanism under which balance is altered in DPN patients under local-control (postural muscle control) and central-control (postural control using sensory cueing). DPN patients and healthy age-matched adults over 55 years performed two 15-second Romberg balance trials. Center of gravity sway was measured using a motion tracker system based on wearable inertial sensors, and used to derive body sway and local/central control balance parameters. Eighteen DPN patients (age = 65.4±7.6 years; BMI = 29.3±5.3 kg/m²) and 18 age-matched healthy controls (age = 69.8±2.9; BMI = 27.0±4.1 kg/m²) with no major mobility disorder were recruited. The rate of sway within local-control was significantly higher in the DPN group by 49% (healthy local-control_slope = 1.23±1.06×10^-2 cm²/sec, P<0.01), which suggests a compromised local-control balance behavior in DPN patients. Unlike local-control, the rate of sway within central-control was 60% smaller in the DPN group (healthy central-control_slope-Log = 0.39±0.23, P<0.02), which suggests an adaptation mechanism to reduce the overall body sway in DPN patients. Interestingly, significant negative correlations were observed between central-control rate of sway with neuropathy severity (r _Pearson = 0.65-085, P<0.05) and the history of diabetes (r _Pearson = 0.58-071, P<0.05). Results suggest that in the lack of sensory feedback cueing, DPN participants were highly unstable compared to controls. However, as soon as they perceived the magnitude of sway using sensory feedback, they chose a high rigid postural control strategy, probably due to high concerns for fall, which may increase the energy cost during extended period of standing; the adaptation mechanism using sensory feedback depends on the level of neuropathy and the history of diabetes.     

Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model

AIM:To investigate the effect of human umbilical cord stem cells,both mesenchymal and hematopoietic(CD34+),in the treatment of arthritis.METHODS:Mesenchymal stem cells(MSCs) and hematopoietic(CD34+) stem cells(HSC) were isolated from human umbilical cord blood obtained from the umbilical cord of healthy pregnant donors undergoing fullterm normal vaginal delivery.MSC,HSC,methotrexate(MTX) and sterile saline were injected intra-articularly into the rat hindpaw with complete freunds adjuvant(CFA) induced arthritis after the onset of disease(day 34),when arthritis had become well established(arthritis score ≥ 2).Arthritic indices were evaluated and the levels of interleukin(IL)-1,tumor necrosis factor(TNF)-α and interferon(IFN)-γ and anti-inflammatory cytokine IL-10 in serum were determined using enzyme-linked immunosorbent assay.Animals of all groups were sacrificed 34 d after beginning treatment,except positive control(PC) which was sacrificed at 10,21 and 34 d for microscopic observation of disease progression.We used hematoxylin,eosin and Masson's trichrome stains for histopathological examination of cartilage and synovium.RESULTS:The mean arthritis scores were similar in all groups at 12 and 34 d post immunization,with no statistical significant difference.Upon the injection of stem cells(hematopoietic and mesenchymal),the overall arthritis signs were significantly improved around 21 d after receiving the injection and totally disappeared at day 34 post treatment in MSC group.Mean hindpaw diameter(mm) in the MSC rats was about half that of the PC and MTX groups(P = 0.007 and P = 0.021,respectively) and 0.6 mm less than the HSC group(P = 0.047),as indicated by paw swelling.Associated with these findings,serum levels of TNF-α,IFN-γ and IL-1 decreased significantly in HSC and MSC groups compared to PC and MTX groups(P 0.05),while the expression of IL-10 was increased.Histopathological examination with H and E stain revealed that the MTX treated group showed significant reduction of leucocytic infiltrate and hypertrophy of the synovial tissue with moderate obliteration of the joint cavity.Stem cells treated groups(both hematopoietic CD34+ and mesenchymal),showed significant reduction in leucocytic infiltrate and hypertrophy of the synovial tissue with mild obliteration of the joint cavity.With Masson's trichrome,stain sections from the PC group showed evidence of vascular edema of almost all vessels within the synovium in nearly all arthritic rats.Vacuoles were also visible in the outer vessel wall.The vessel became hemorrhagic and finally necrotic.In addition,there was extensive fibrosis completely obliterating the joint cavity.The mean color area percentage of collagen in this group was 0.324 ± 0.096,which was significantly increased when compared to the negative control group.The mean color area percentage of collagen in hematopoietic CD34+ and mesenchymal groups was 0.176 ± 0.0137 and 0.174 ± 0.0197 respectively,which showed a marked decrement compared to the PC group,denoting a mild increase in synovial tissue collagen fibers.CONCLUSION:MSC enhance the efficacy of CFAinduced arthritis treatment,most likely through the modulation of the expression of cytokines and amelioration of pathological changes in joints.     

Improvement in infected wound healing in type 1 diabetic rat by the synergistic effect of photobiomodulation therapy and conditioned medium

We investigated the effects of photobiomodulation therapy (PBMT) and conditioned medium (CM) of human bone marrow mesenchymal stem cells (hBM-MSC) individually and/or in combination on the stereological parameters and the expression of basic fibroblast growth factor (bFGF), hypoxia-inducible factor (HIF-1α), and stromal cell–derived factor-1α (SDF-1α) in a wound model infected with methicillin-resistant Staphylococcus aureus (MRSA) in diabetic rats. CM was provided by culturing hBM-MSCs. Type 1 diabetes mellitus (T1DM) was induced in 72 rats, divided into four groups, harboring 18 rats each: group 1 served as a control group, group 2 received PBMT, group 3 received CM, and group 4 received CM + PBMT. On days 4, 7, and 15, six animals from each group were euthanized and the skin samples were separated for stereology examination and gene expression analysis by real-time polymerase chain reaction. In the CM + PBMT, CM, and PBMT groups, significant decreases were induced in the number of neutrophils (1460 ± 93, 1854 ± 138, 1719 ± 248) and macrophages (539 ± 69, 804 ± 63, 912 ± 41), and significant increases in the number of fibroblasts (1073 ± 116, 836 ± 75, 912 ± 41) and angiogenesis (15 230 ± 516, 13 318 ± 1116, 14 041 ± 867), compared with those of the control group (2690 ± 371, 1139 ± 145, 566 ± 90, 12 585 ± 1219). Interestingly, the findings of the stereological examination in the CM + PBMT group were statistically more significant than those in the other groups. In the PBMT group, in most cases, the expression of bFGF, HIF-1α, and SDF-1α, on day 4 (27.7 ± 0.14, 28.8 ± 0.52, 27.5 ± 0.54) and day 7 (26.8 ± 1.4, 29.6 ± 1.4, 28.3 ± 1.2) were more significant than those in the control (day 4, 19.3 ± 0.42, 25.5 ± 0.08, 22.6 ± 0.04; day 7, 22.3 ± 0.22, 28.3 ± 0.59, 24.3 ± 0.19) and other treatment groups. The application of PBMT + CM induced anti-inflammatory and angiogenic activities, and hastened wound healing process in a T1 DM model of MRSA infected wound.     

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.     

Let-7e enhances the radiosensitivity of colorectal cancer cells by directly targeting insulin-like growth factor 1 receptor

Abnormal expression of various microRNAs (miRNAs), as regulators of biological signaling pathways, has a strong association with cancer resistance to chemotherapy and radiotherapy. The let-7 family of miRNAs as tumor suppressors have shown to be downregulated in different types of human malignancies including colorectal cancer (CRC). However, the biological function of let-7 members in the processes of resistance to radiation in CRC has not yet been completely elucidated. Insulin-like growth factor 1 receptor (IGF-1R) signaling pathway is amplified in CRC and leads to its progression, development, and also radiation resistance. So, it seems like an attractive target for anticancer therapy. In this study, by using bioinformatics analysis, it has been revealed that IGF-1R is a direct target of the let-7e member. Consistent with this, we identified that increased levels of let-7e in CRC cells reduced IGF-1R protein level and subsequently its downstream signaling pathways, which resulted in the G1 cell cycle arrest and a significant reduction in the proliferation, survival and also resistance to radiation of CRC cells. Altogether, these results suggested that let-7e by targeting the IGF-1R signaling pathway might serve as therapeutics in anticancer therapy.     

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity.

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.