Transient Low-Temperature Effects on Propidium Iodide Uptake in Lance Array Nanoinjected HeLa Cells

2015 ◽  
Vol 6 (4) ◽  
Author(s):  
John W. Sessions ◽  
Brad W. Hanks ◽  
Dallin L. Lindstrom ◽  
Sandra Hope ◽  
Brian D. Jensen
Keyword(s):  
Propidium Iodide ◽  
Positive Control ◽  
P Value ◽  
Acid Dye ◽  
Iodide Uptake ◽  
Maximum Value ◽  
Nonviral Transfection ◽  
A Cell ◽  
Effects Of Temperature ◽  
Lance Array Nanoinjection

Understanding environmental factors relative to transfection protocols is key for improving genetic engineering outcomes. In the following work, the effects of temperature on a nonviral transfection procedure previously described as lance array nanoinjection are examined in context of molecular delivery of propidium iodide (PI), a cell membrane impermeable nucleic acid dye, to HeLa 229 cells. For treatment samples, variables include varying the temperature of the injection solution (3C and 23C) and the magnitude of the pulsed voltage used during lance insertion into the cells (+5 V and +7 V). Results indicate that PI is delivered at levels significantly higher for samples injected at 3C as opposed to 23C at four different postinjection intervals (t = 0, 3, 6, 9 mins; p-value ≤ 0.005), reaching a maximum value of 8.3 times the positive control for 3 C/7 V pulsed samples. Suggested in this work is that between 3 and 6 mins postinjection, a large number of induced pores from the injection event close. While residual levels of PI still continue to enter the treatment samples after 6 mins, it occurs at decreased levels, suggesting from a physiological perspective that many lance array nanoinjection (LAN) induced pores have closed, some are still present.

scholarly journals Inhibitory Effects of Secondary Metabolites from the Lichen Stereocaulon evolutum on Protein Tyrosine Phosphatase 1B

Planta Medica ◽  
2021 ◽  
Author(s):  
Birgit Waltenberger ◽  
Françoise Lohézic-Le Dévéhat ◽  
Thi Huyen Vu ◽  
Olivier Delalande ◽  
Claudia Lalli ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Inhibitory Activity ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Positive Control ◽  
Protein Tyrosine Phosphatase 1B ◽  
Protein Tyrosine ◽  
Ic50 Values ◽  
A Cell

AbstractProtein tyrosine phosphatase 1B plays a significant role in type 2 diabetes mellitus and other diseases and is therefore considered a new drug target. Within this study, an acetone extract from the lichen Stereocaulon evolutum was identified to possess strong protein tyrosine phosphatase 1B inhibition in a cell-free assay (IC50 of 11.8 µg/mL). Fractionation of this bioactive extract led to the isolation of seven known molecules belonging to the depsidones and the related diphenylethers and one new natural product, i.e., 3-butyl-3,7-dihydroxy-5-methoxy-1(3H)-isobenzofurane. The isolated compounds were evaluated for their inhibition of protein tyrosine phosphatase 1B. Two depsidones, lobaric acid and norlobaric acid, and the diphenylether anhydrosakisacaulon A potently inhibited protein tyrosine phosphatase 1B with IC50 values of 12.9, 15.1, and 16.1 µM, respectively, which is in the range of the protein tyrosine phosphatase 1B inhibitory activity of the positive control ursolic acid (IC50 of 14.4 µM). Molecular simulations performed on the eight compounds showed that i) a contact between the molecule and the four main regions of the protein is required for inhibitory activity, ii) the relative rigidity of the depsidones lobaric acid and norlobaric acid and the reactivity related to hydrogen bond donors or acceptors, which interact with protein tyrosine phosphatase 1B key amino acids, are involved in the bioactivity on protein tyrosine phosphatase 1B, iii) the cycle opening observed for diphenylethers decreased the inhibition, except for anhydrosakisacaulon A where its double bond on C-8 offsets this loss of activity, iv) the function present at C-8 is a determinant for the inhibitory effect on protein tyrosine phosphatase 1B, and v) the more hydrogen bonds with Arg221 there are, the more anchorage is favored.

scholarly journals In vivo antidiarrheal study of ethanolic extracts of Mikania cordata and Litsea monopetala leaves

2015 ◽  
Vol 10 (3) ◽  
pp. 562 ◽  
Author(s):  
Fatema Nasrin ◽  
Md. Lukman Hakim
Keyword(s):  
Body Weight ◽  
Castor Oil ◽  
Leaf Extract ◽  
Positive Control ◽  
Control Group ◽  
P Value ◽  
Leaf Extracts ◽  
Ethanolic Extracts ◽  
Antidiarrheal Activity

<p class="Abstract">In this study the antidiarrheal activity of ethanolic extracts of the leaves of <em>Mikania cordata</em> and <em>Litsea monopetala</em> was evaluated. Diarrhea was induced in mice by oral administration of castor oil (0.5 mL) 30 min after the administration of the extracts. During a 4 hour study the number of diarrheal feces and percentage inhibition of the extracts (200 and 400 mg/kg body weight) was determined. Loperamide (3 mg/kg body weight) served as standard and belonged to the positive control group. The extracts exhibited potent antidiarrheal activity as well as achieved statistically significant p value (p&lt;0.01 and p&lt;0.05) compared to control group. Among the extracts the highest percentage inhibition of defecation (60%) was recorded for leaf extract (400 mg/kg body weight) of <em>L. monopetala</em>. So, the study corroborates the significant antidiarrheal activity of <em>M. cordata</em> and <em>L. monopetala</em> leaf extracts and raises the demand of further sophisticated investigation.</p><p> </p>

scholarly journals Photobiomodulation of fresh bone marrow aspirate for regenerative therapy

2021 ◽  
Vol 10 (11) ◽  
pp. e140101119545
Author(s):  
Carolina dos Santos Santinoni ◽  
Liziana Jancos Calles ◽  
Nathália Laís Farias ◽  
Thaís Sanches Leite Patara ◽  
Bianca Eduarda de Lima Neves ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Aspirate ◽  
Positive Control ◽  
Negative Control ◽  
Low Level Laser Therapy ◽  
Adult Rats ◽  
Level Laser ◽  
Group Water ◽  
A Cell ◽  
Fresh Bone

Use of mesenchymal stem cells and low-level laser therapy (LLLT) have been widely studied to promote bone healing. evaluate effect of photobiomodulation on total number of cells (TNC) and cell viability (CV) of fresh bone marrow aspirate (BMA). Femur BMA from 10 adult rats was collected and a cell concentration of 1x107 cell/mL was obtained. Cell suspension was deposited on 96 well cell culture plates and distributed in groups: 1) RPMI, positive control; 2) Distilled Water, negative control; 3) Red Laser (RL); 4) Infrared Laser (IRL). Groups RL and IRL received LLLT application right after incubation. Cells were incubated for 24 h. TNC and CV were assessed through trypan blue assay after 1, 3, 6, 10 and 24 h of incubation. Data distribution was verified by Shapiro-Wilk test. Kruskal-Wallis test was used for intergroup and intragroup comparisons (p<0.05). TNC: after 1 and 3 h, groups RL and IRL presented significantly higher TNC than Group Water; after 6 and 10 h, groups RPMI, RL and IRL presented significantly higher TNC than Group Water. CV: after 1 h, groups RL and IRL showed significantly higher percentage of VC than Group Water; after 3, 6 and 10 h, all groups presented significantly higher percentage of VC than Group Water. It can be concluded that LLLT enhanced number and viability of fresh bone marrow aspirate cells.

scholarly journals THE EFFECT OF RECOMBINANT VASCULAR ENDOTHELIAL GROWTH FACTOR 121 ON NITRIDE OXIDE LEVEL IN MICE (Mus musculus) MODEL OF PREECLAMPSIA

2017 ◽  
Vol 53 (3) ◽  
pp. 191
Author(s):  
Soetrisno Soetrisno ◽  
Isharyadi Isharyadi ◽  
Sri Sulistyowati
Keyword(s):  
Nitric Oxide ◽  
Mus Musculus ◽  
P Value ◽  
Vascular Endothelial ◽  
The Third ◽  
Minimum Value ◽  
Maximum Value ◽  
Pregnant Mice ◽  
Endothelial Growth Factor ◽  
K2 Group

Preeclampsia is a multifactorial syndrome in pregnancy whose cause is still unknown. Several proangiogenic and antiangiogenic mediators such as Vascular Endothelial Growth Factor (VEGF) and Nitrite Oxide (NO) play important roles in preventing preeclampsia. VEGF can increase NO level that lowers maternal blood pressure, improves endothelial function and reduces placental hypoxia in preeclampsia. Recombinant VEGF 121 is expected to be an option in the prevention and treatment of preeclampsia. This experimental study used mice (Mus musculus) as the model. The objective of this study was to observe the effect of recombinant VEGF 121 in increasing the level of nitric oxide in mice (Mus musculus) model of preeclampsia. This was an experimental analytical study with Randomized Control Trial (RCT) design. The study enrolled 27 pregnant mice (Mus musculus) which met the restriction criteria divided into 3 groups. The first group (K1) were 9 normal pregnant mice. The second group (K2) were 9 pregnant mice of preeclampsia model without treatment. The third group (K3) were 9 pregnant mice of preeclampsia model receiving recombinant VEGF 121 therapy. The independent variable was the administration of recombinant VEGF 121 and the dependent variable was the serum NO level. Statistical analysis was performed by using anova statistics. NO level in the first group (K1) was 1.746±0.347, with minimum value of 1.00 µM, and maximum value of 2.28 µM, CI (1.479-2.013).  NO level in second group (K2) was 1.167±0.380, with minimum value of 0.64 µM, and maximum value of 1.94 µM, CI (0.875-1.460). NO level in the third group (K3) was 2.164±0.556, with minimum value of 1.56 µM, and maximum value of 5.96 µM, CI (1.842-2.486). With anova statistical test, there were significant differences between K1 group and K2 group (p value=0.004<0.05), K1 group and K3 group (p value=0.000<0.05) as well as K2 group and K3 group (p value=0.029<0.05). In conclusion, Recombinant VEGF 121 increased the level of nitric oxide in mice (Mus musculus) model of preeclampsia significantly.

scholarly journals Reported History of Measles and Long-term Impact on Antibody to Tetanus in Children 6–59 Months of Age Receiving DTP in the Democratic Republic of Congo

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S323-S323 ◽  
Author(s):  
Hayley Ashbaugh ◽  
James D Cherry ◽  
Sue Gerber ◽  
Stephen G Higgins ◽  
Adva Gadoth ◽  
...  
Keyword(s):  
Democratic Republic ◽  
Democratic Republic Of Congo ◽  
Past History ◽  
Positive Control ◽  
P Value ◽  
Republic Of Congo ◽  
History Of ◽  
Long Term Impact ◽  
Induced Immunity

Abstract Background Recent studies suggest a measles-induced immune amnesia that could have long-term immunosuppressive effects via preferential depletion of memory B and T CD150+ lymphocytes. Methods We examined the association between past measles and tetanus antibody levels among children participating in the 2013–2014 Democratic Republic of Congo (DRC) Demographic and Health Survey (DHS). Our sample consisted of 833 children aged 6–59 months whose mothers were selected for interview. Mothers reported (via recall) history of measles within the lifetime of the child. Classification of children who previously had measles was completed using maternal recall and measles immunoglobulin G (IgG) serostatus obtained via dried blood spot (DBS) analysis. A multiplex chemiluminescent immunoassay platform was used to obtain serologic results and Assay Score (AS) was calculated as a ratio to a positive control included in each run. Tetanus serostatus was categorized as being above or below the sample median serology AS value. Tetanus vaccination status was obtained via dated vaccination card and limited to children receiving the complete 3-dose vaccination series. Results The median AS for tetanus serology among the entire sample of 833 children was 0.085, while children with history of measles had a median AS of 0.053 (N = 41) and children with no history of measles had a median AS of 0.088 (N = 792), chi-square P-value &lt; 0.05. A random intercept logistic regression model was used to examine the association between previous measles disease and odds of having below median levels of tetanus antibody. Controlling for potential confounding variables, the odds of a child with past history of measles having less than the median level of tetanus antibody was 3.86 (95% CI: 1.70, 8.78) among children fully vaccinated for tetanus. Conclusion The results suggest that, among children 6–59 months in DRC, measles may have a long-term impact on levels of pre-existing, vaccine-induced immunity to tetanus. These findings suggest the need for laboratory studies examining measles’ impact on pre-existing, vaccine-induced immunity and underscore the need for continued evaluation and improvement of DRC’s measles vaccination program. Disclosures All authors: No reported disclosures.

Influence of Temperature Differential between Tomatoes and Postharvest Water on Salmonella Internalization

2016 ◽  
Vol 79 (6) ◽  
pp. 922-928 ◽  
Author(s):  
ASHLEY N. TURNER ◽  
LORETTA M. FRIEDRICH ◽  
MICHELLE D. DANYLUK
Keyword(s):  
Abscission Zone ◽  
Most Probable Number ◽  
P Value ◽  
Temperature Differential ◽  
Time Intervals ◽  
Probable Number ◽  
Treatment Conditions ◽  
Pulp Temperature ◽  
Tomato Pulp ◽  
Effects Of Temperature

ABSTRACT Salmonella bacteria may internalize into tomato pulp when warm tomatoes from the field are submerged into colder water. Several washing steps may follow the initial washing and packing of tomatoes at the packinghouses; the potential for internalization into tomatoes in subsequent washing steps when tomatoes have a cooler pulp temperature is unknown. Our objective was to evaluate Salmonella internalization into mature green and red tomatoes with ambient (21°C) and refrigeration (4°C) pulp temperatures when they were submerged into water at various temperature differentials, simulating repacking and fresh-cut operations. Red (4°C and 21°C) and mature green (21°C) tomatoes were submerged (6 cm) into a six-strain Salmonella cocktail (6 log CFU/ml) and maintained at ±5 and 0°C temperature differentials for varying time intervals, ranging from 30 s to 5 min. Following submersion, tomatoes were surface sterilized using 70% ethanol, the stem abscission zone and blossom end epidermis were removed, and cores were recovered, separated into three segments, and analyzed. Salmonella populations in the segments were enumerated by most probable number (MPN). The effects of temperature differential and maturity on Salmonella populations were analyzed; results were considered significant at a P value of ≥0.5. Internalized populations were not significantly different (P ≥ 0.5) across temperature differentials. Salmonella internalization was seen in tomatoes under all treatment conditions and was highest in the segment immediately below the stem abscission zone. However, populations were low (typically &gt;1 log MPN per segment) and varied greatly across temperature differentials. This suggests that the temperature differential between tomatoes and water beyond the initial packinghouse may be less important than submersion time in Salmonella internalization.

Effects of 1% and 3% Mobe Leaf Extract Gel on Socket Wound Healing after Tooth Extraction

2021 ◽  
Vol 24 (1) ◽  
pp. 1-5
Author(s):  
Olivia Avriyanti Hanafiah ◽  
Denny Satria ◽  
Avi Syafitri
Keyword(s):  
Wound Healing ◽  
Tooth Extraction ◽  
Leaf Extract ◽  
Alveolar Bone ◽  
Negative Control Group ◽  
Positive Control ◽  
Negative Control ◽  
Control Group ◽  
P Value ◽  
Gel Treatment

Tooth extraction is a process of removing teeth from the alveolar bone. In wound healing, fibroblast are very important cells. The main purpose of this study was to determine the effect of mobe leaf 1% and 3% extract gel (Artocarpus lakoocha) on fibroblast proliferation in post extraction tooth socket wound healing. This research used 16 samples of wistar rats, divided into 4 groups, a positive control group, a negative control group and a 1% and 3% mobe leaf extract gel group. The left mandibular incisors were extracted, then 1% and 3% gels of mobe leaf extract were applied on day 1 to day 7. Data analysis was calculated using the Kruskal-Wallis test on clinical data and one way ANOVA test for microscopic. The result of the socket wound healing activity test for a good concentration of mobe leaf extract gel was 3%. This research shows significant resultith p-value of 0.018 (< 0.05) on the closure of the socket wound clinically which means the closure of the wound accelerates because of the mobe leaf 3% extract gel treatment. The distance of fibroblast on microscopically shows significant resultith a p-value of 0.002 (< 0.05), which means that there was an enlargement of the distance fibroblast at the socket wound closure with application of mobe leaf 3% extract gel. From the results of the study it can be concluded that mobe leaf 3% extract gel has the best ability to show acceleration the closure of the socket wound either clinically or microscopically.

scholarly journals AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating.

1989 ◽  
Vol 9 (8) ◽  
pp. 3155-3165 ◽  
Author(s):  
P N Lipke ◽  
D Wojciechowicz ◽  
J Kurjan
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Structural Gene ◽  
Signal Sequence ◽  
Positive Control ◽  
Complementation Group ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Amino Terminal ◽  
A Cell

We have cloned the alpha-agglutinin structural gene, AG alpha 1, by the isolation of alpha-specific agglutination-defective mutants, followed by isolation of a complementing plasmid. Independently isolated alpha-specific agglutination-defective mutations were in a single complementation group, consistent with biochemical results indicating that the alpha-agglutinin is composed of a single polypeptide. Mapping results suggested that the complementation group identified by these mutants is allelic to the ag alpha 1 mutation identified previously. Expression of AG alpha 1 RNA was alpha specific and inducible by a-factor. Sequences similar to the consensus sequences for positive control by MAT alpha 1 and pheromone induction were found upstream of the AG alpha 1 initiation codon. The AG alpha 1 gene could encode a 650-amino-acid protein with a putative signal sequence, 12 possible N-glycosylation sites, and a high proportion of serine and threonine residues, all of which are features expected for the alpha-agglutinin sequence. Disruption of the AG alpha 1 gene resulted in failure to express alpha-agglutinin and loss of cellular agglutinability in alpha cells. An Escherichia coli fusion protein containing 229 amino acids of the AG alpha 1 sequence was recognized by an anti-alpha-agglutinin antibody. In addition, the ability of this antibody to inhibit agglutination was prevented by this fusion protein. These results indicate that AG alpha 1 encodes alpha-agglutinin. Features of the AG alpha 1 gene product suggest that the amino-terminal half of the protein contains the a-agglutinin binding domain and that the carboxy-terminal half contains a cell surface localization domain, possibly including a glycosyl phosphatidylinositol anchor.

scholarly journals Autoantibodies from patients with autoimmune thyroid disease do not interfere with the activity of the human iodide symporter gene stably transfected in CHO cells

2001 ◽  
pp. 611-618 ◽  
Author(s):  
M Tonacchera ◽  
P Agretti ◽  
G Ceccarini ◽  
R Lenza ◽  
S Refetoff ◽  
...  
Keyword(s):  
Cell Line ◽  
Cho Cells ◽  
Hashimoto’S Thyroiditis ◽  
Time Course ◽  
Hashimoto's Thyroiditis ◽  
Graves Disease ◽  
Dependent Manner ◽  
Iodide Uptake ◽  
Autoimmune Thyroid ◽  
A Cell

OBJECTIVE: The human sodium iodide symporter (hNIS) is a candidate autoantigen in autoimmune thyroid diseases. To investigate the possible existence of autoantibodies able to interfere with the biological activity of hNIS, an assay was developed using a cell line stably expressing hNIS. METHODS: hNIS complementary cDNA cloned in pcDNA3 and a neomycin resistance gene vector were co-transfected into CHO cells. After selection with geneticin, a cell line termed PA4, showing the highest level of Na(125)I uptake, was characterized. The time course of iodide uptake was evaluated by incubating PA(4) cells with 10 micromol/l NaI and 0.1 microCi Na(125)I for a period up to 90 min. The accumulation of iodide increased linearly between 2 and 10 min, reaching a plateau at 45 min. The curve of iodide efflux mirrored that of iodide influx. Both perchlorate and thiocyanate inhibited iodide uptake in PA(4) cells in a dose-dependent manner starting from concentrations as low as 0.01 and 0.1 micromol/l respectively and complete inhibition was obtained at concentrations of 100 micromol/l perchlorate and 1000 micromol/l thiocyanate. The sensitivity of the inhibition assay was further improved using both inhibitors after 5 min incubation and in the absence of cold NaI. RESULTS: Included in the study were 42 patients with Graves' disease (25 had active hyperthyroidism, ten were euthyroid and seven had hypothyroidism); 34 patients with Hashimoto's thyroiditis (one was euthyroid, four had subclinical hypothyroidism and 29 were overtly hypothyroid); and 19 with atrophic thyroiditis (all hypothyroid). Four out of eight whole sera from patients with Hashimoto's thyroiditis, and 8 out of 25 whole sera from patients with Graves' disease caused an inhibition of iodide uptake in PA(4) cells greater than 20% but also in 4 out of 15 sera from normal subjects. This inhibition activity exerted by sera from patients and controls was lost after dialyzing against buffer. Accordingly, IgGs purified from sera of all patients with Graves' disease and with Hashimoto's thyroiditis or atrophic thyroiditis were devoid of any effect on iodide uptake. CONCLUSIONS: In conclusion, we believe that autoantibodies able to block the function of hNIS are very rare.

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