scholarly journals Sonication and Short-term Incubation Alter the Content of Bovine Milk Exosome Cargos and Exosome Bioavailability (OR26-08-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Sonal Sukreet ◽  
Bruno Vieira Resende E Silva ◽  
Jiri Adamec ◽  
Juan Cui ◽  
Janos Zempleni

Abstract Objectives Exosomes are endogenous nanoparticles that participate in cell-to-cell communication through the transfer of cargos such RNAs, lipids and proteins from donor cells to recipient cells. Previously, we showed that mammals absorb exosomes from milk. Ultrasonication causes a transient disruption of the exosome membranes, leading to loss of microRNAs. When mice were fed diets based on the AIN-93G formulation, modified to contain a physiological amount of milk exosomes (exosome and RNA-sufficient diet, ERS) or sonicated exosomes (exosome and RNA-depleted diet, ERD), we observed a loss of circulating and tissue microRNAs and phenotypes such as aberrant purine metabolism. The objective of this study was to conduct a comprehensive analysis ofthe effects of sonication on exosomes cargos and bioavailability, thereby generating insights into mechanisms through which ERD elicits phenotypes. Methods Exosomes were isolated from ultrasonicated (USE) and non-sonicated (NSE) bovine milk by ultracentrifugation and authenticated following guidelines of the International Society for Extracellular Vesicles. MicroRNAs were analyzed by small RNA-sequencing. Lipids and proteins were analyzed by LC/MS-MS. Intestinal transport was assessed using FM 4-64-labeled exosomes in primary human small intestine cells (FHs cells). Bioavailability of exosomes transfected with IRDye-labeled miR-320a was assessed using oral gavage in C57BL/6 mice.The unpaired t-test was used for statistical analysis P < 0.05 was considered statistically significant. Results Ultrasonication affected the vesicle count and exosome morphology. Western blot analysis detected marker proteins only in NSE. The content of microRNAs was about 93% lower in USE than NSE. Significant difference was noted for lipid and protein identities between NSE and USE. Reduced uptake of USE by intestinal cells and loss of cargo accumulation in murine livers and pancreas for USE compared to NSE (Fig. 1-5). Conclusions Ultrasonication causesa loss of microRNAs in milk exosomes. The unique patterns of proteins and lipids likely is due to an exchangeof membranes between exosomes and other vesicles during ultrasonication, which might explain the lower bioavailability of USE compared to NSE. We currently test the exchange of lipids during ultrasonication. Funding Sources NIFA, NIH, Gates Foundation, PureTech, Inc. and USDA Hatch and Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Di Wu ◽  
Jiang Shu ◽  
Camila Braga Pereira ◽  
Juan Cui ◽  
Jiri Adamec ◽  
...  

Abstract Objectives Exosomes are natural nanoparticles that participate in cell-to-cell communication by transferring regulatory molecules such as microRNAs from donor cells to recipient cells. Previously, we have demonstrated that exosomes and microRNAs are not exclusively obtained from endogenous synthesis but may also be absorbed from milk. We assessed whether 1) exosomes contain mRNAs (i.e., RNAs other than microRNAs), 2) mRNAs are bioavailable in mice, and 3) bovine mRNAs are translated into peptides. Methods For mRNA analysis, exosomes were isolated from bovine milk using differential ultracentrifugation, and RNA cargos were analyzed by RNA-sequencing. For bioavailability analysis, a synthetic fragment of fluorophore (IRDye)-labeled bovine CSN3 mRNA was transfected into bovine milk exosomes, which were administered to Balb/c mice by oral gavage. Tissues distribution of CSN3 mRNA was assessed 24 h after gavage by using a LiCor Odyssey CLx imager. For analysis of mRNA translation, mRNA from bovine milk exosomes were translated using the rabbit reticulocyte lysate system and BODIPY-labeled lysine. Peptides were separated by two-dimensional gel electrophoresis and fluorescence was visualized using a Typhoon FLA 7000 scanner. Statistical analysis, NA. Results We detected > 3600 bovine mRNAs in exosomes. Most mRNAs were truncated; 107 mRNAs contained their natural ATG start codons. Thirteen of these mRNAs, including CSN3, encoded bovine proteins and peptides with amino acid sequences distinct from those in human and murine orthologs (Table 1). Mice absorbed CSN3 mRNA encapsulated in milk exosomes, which accumulated primarily in the liver (Fig. 1). Nine bovine peptide spots were detected by two-dimensional electrophoresis (Fig. 2). Conclusions Bovine milk exosomes contain mRNAs which are bioavailable and translated into peptides in non-bovine systems. We speculate that food mRNAs might play a role in food allergies and immune tolerance. Future studies: Identification of peptides by LC/MS-MS is in progress. We will assess the relevance of mRNA translation for allergies and tolerance in animal models. Funding Sources NIFA, NIH, Gates Foundation, PureTech, Inc. and USDA Hatch and Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs


2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 946-946
Author(s):  
Mojisola Ogunnaike ◽  
Janos !Zempleni

Abstract Objectives Exosomes are natural nanoparticles that play an important role in cell-to-cell communication. Communication is achieved through the binding of exosomes to the surface of recipient cells or delivery of regulatory cargos such as microRNAs (miRs) to recipient cells. Exosomes can be detected in most body fluids including milk. We reported that exosomes and miR cargos do not originate exclusively in endogenous synthesis but humans and other mammals absorb BMEs. The field of BME bioavailability is somewhat controversial and would benefit from the availability of a tool that facilitates BME tracking in organisms. The objective of this study was to compare the characteristics of BMEs and exosomes secreted by Mac-T cells as a step toward using Mac-T cell exosomes in future studies of BME bioavailability and distribution. Methods Exosomes were isolated from Mac-T conditioned culture media and skim milk by using ultracentrifugation. Exosome count, size, morphology, and purity were assessed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and immunoblot analysis, respectively. MiR cargos were assessed by qPCR. Levene's test, Shapiro-Wilk test, and unpaired t-test were applied for statistical analysis; p &lt; 0.05 was considered statistically significant. Results Morphology (not shown) and size were similar in BMEs (101 + 7.6 nm) and Mac-T derived exosomes (94 ± 10 nm; n = 3, p &gt; 0.05). Exosome marker proteins, CD9, CD63 and TSG101, were detected in both BMEs and Mac-T cell-derived exosomes. Likewise, both preparations expressed miR-320a, miR-200c and let7a (p &gt; 0.05). Conclusions Mac-T cell-derived exosomes have characteristic similar to BMEs and may be a useful tool in studies of BME bioavailability and distribution. Funding Sources NIH 1P20GM104320, NIFA 2016-67,001-25,301 and 2020-67,017-30,834, USDA Hatch-1,011,996 and W4002 (all to J.Z.). J.Z is a consultant for PureTech Health, Inc.


2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1113-1113
Author(s):  
Fang Zhou ◽  
Janos Zempleni

Abstract Objectives Exosomes play a role in cell-to-cell communication, which is achieved by the transfer of microRNAs from donor cells to recipient cells. Exosomes and their microRNA cargos may also be absorbed from milk and accumulate primarily in the intestinal mucosa, kidneys and brains in C57BL/6 pups. MicroRNA biogenesis depends on the endonuclease Dicer, and homozygous Dicer KO is lethal. We hypothesized maternal microRNA biogenesis would be important for optimal postnatal development of pups. This study aimed to assess whether maternal loss of microRNA biogenesis impairs the nutritional quality of milk and gut health in wild-type (WT) pups fostered to Dicer knockout (KO) dams. Methods WT C57BL/6 pups were fostered to tamoxifen-inducible heterozygous Dicer KO dams from synchronized pregnancies (4 pups/dam). In the treatment group, one allele of Dicer was knockout out in dams by i.p. tamoxifen injection after delivery (denoted “Dicer KO”); control dams were injected with vehicle (denoted “CTRL”). Milk was collected 11 days postpartum for analysis of exosomal microRNAs (RT-qPCR), lactose (enzymatic), total protein (BCA assay), total triglycerides (Vitros 250 Analyzer) and water (weighing oven). Postnatal gut morphology and gut barrier function were assessed by histology analysis and oral FITC-dextran at age 3 weeks. The unpaired t-test was used for statistical analysis; P &lt; 0.05 was considered statistically significant. Results The expression of microRNAs in exosomes decreased by 60% in Dicer KO dams compared to CTRL. The loss of maternal microRNA biogenesis impaired gut health in WT pups: 1) The length of the gastrointestinal tract and the width of jejunum decreased by more than 33% and 41% in WT pups fostered to Dicer KO dams, respectively, compared to pups fostered to CTRL. 2) The villi height decreased by 20% (female) and 10% (male) in WT pups fostered Dicer KO dams compared to pups fostered to CTRL. 3) Gut barrier function was impaired, as evidenced by a peak FITC-dextran plasma concentration that was 59% higher in pups fostered to Dicer KO compared to CTRL after oral FITC-dextran. Dicer KO had no effect on the content of macronutrients and water in milk. Conclusions MicroRNAs in milk play a role in optimal postnatal gut health during lactation. Funding Sources NIFA, NIH, Gates Foundation and USDA. J.Z. is a consultant for PureTech Health.


2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Sonal Sukreet ◽  
Bruno Vieira Resende E Silva ◽  
Jiri Adamec ◽  
Juan Cui ◽  
Janos Zempleni

Abstract Objectives Exosomes are natural nanoparticles that can be found in most body fluids including milk. Bovine milk exosomes (BME) are bioavailable and transfer cargos such as RNAs, lipids and proteins across species boundaries. Previous competitor studies implicated galactose-modifications in exosome surface proteins in exosome transport. The objectiveof our study was to assess the importance of glycan modifications in BME surface glycoproteins for BME uptake and distribution in human intestinal cells (FHs cells) and mice. Methods BME were isolated from milk by differential centrifugation (Fig. 1). Putative glycan binding sites on the surfaces of BME were identified by LC/MS-MS and bioinformatics protocols and confirmed by eastern blotting. Surface glycans were altered using proteases, glycosidases and blocking of glycans with lectins for subsequent transport and distribution studies (Table 1). The uptake of BME by FHs cells was assessed using membrane and RNA cargo-labeled BME. The distribution of BME in mice was assessed using BME transfected with fluorophore (IRDye)-labeled synthetic miR-320a in C57BL/6 mice and MGAT-1 tamoxifen inducible conditional knockout (CKO) mice ages 5–8 weeks. One-way ANOVA and Bonferroni's multiple comparison were used for statistical analyses; P < 0.05 was considered significant. Results We identified 49 (N), 13 (O) and 13 (C) putative glycan binding sites on the BME surface, and confirmed the presence of mannose, galactose, N-acetylgalactose, fucose and neuraminate (Fig. 2). Galactose and N-acetylgalactosamine residues on the surface of BME were of particular importance for their uptake by FHs cells, whereas N-acetylglucosamine on the surface of FHs cells was more important than other glycans for BME uptake (Fig. 3). Enzymatic and genetic removal of glycans in BME and MGAT-1 CKO, respectively, caused a loss of BME accumulation in murine livers and pancreas (Fig. 4). Conclusions Distinct glycan features are essential for the uptake of BME in mice. The study laid a foundation to assess the importance of glycan modifications in infant formulas and the homing of drug-loaded exosomes to sites of disease. Funding Sources NIFA, NIH, Bill & Melinda Gates Foundation, PureTech Health, and USDA Hatch & Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs


2020 ◽  
Vol 73 (4) ◽  
pp. 148-152
Author(s):  
Kornél Vajda ◽  
László Sikorszki

Összefoglaló. Bevezetés: A laparoszkópia térhódítása a jobb oldali colon műtéteknél is nyilvánvaló. Ma legtöbb helyen a laparoszkóposan asszisztált jobb oldali hemikolektómia extrakorporális anasztomózissal a gold standard. A morbiditás randomizált vizsgálatok alapján még 30% körüli. A technikai fejlődés lehetővé tette az intrakorporális anasztomózist. Célkitűzés: Retrospektív módon elemezni rosszindulatú jobb oldali vastagbéldaganat miatt végzett laparoszkópos hemikolektómiák rövid távú eredményeit a két módszer összehasonlításával. Eredmények: 2018. 01. 01. – 2019. 12. 31. között 184 jobb oldali hemikolektómiát végeztünk, ezek közül 122 történt malignus betegség miatt. 51 esetben nyitott és 71 esetben laparoszkópos műtét történt. 37 férfi (átlagéletkor: 70,59 év) és 34 nő (átlagéletkor: 72,14 év) volt. 50 esetben extrakorporális (EA) és 21 esetben pedig intrakorporális anasztomózist (IA) végeztünk. Az EA csoportban 18, míg az IA csoportban 3 szövődmény alakult ki 30 napon belül (p = 0,067). Az EA csoportból 3, az IA csoportból 1 beteget veszítettünk el 30 napon belül (p = 0,66). Az átlagos ápolási idő az EA csoportban 9,48 (5–32) nap, míg az IA csoportban 6,52 (4–19) nap volt (p = 0,001) a szövődményes esetekkel együtt. A szövődményes esetek nélkül az EA csoportban 6,35 (5–10) nap, az IA csoportban pedig 5,55 (4–8) napnak bizonyult (p = 0,09). A műtéti idő pedig az EA csoportban 147 (90–240) perc, az IA csoportban pedig 146,47 (90–265) perc volt (p = 0,11). Konklúzió: Az irodalommal összhangban azt találtuk, hogy IA esetén kevesebb a szövődmény, ezzel is összefüggésben rövidebb az átlagos ápolási idő, és a műtéti időt tekintve nincs szignifikáns különbség. Ezeket figyelembe véve az intrakorporális anasztomózis javasolható jobb oldali laparoszkópos hemikolektómia esetén. Summary. Introduction: Laparoscopy became evident for right-sided colon surgery too. Today the laparoscopic-assisted right-hemicolectomy is the gold standard with extracorporeal anastomosis. Morbidity according to randomized trials is still approximately 30%. The development of the surgical technique resulted in the creation of intracorporeal anastomosis. Our aim was to compare the short-term results of the two methods. Aim: To analyse the short-term results of right-sided hemicolectomy that were performed due to malignant tumours with the comparison of the two methods. Results: A cohort of 184 right-sided hemicolectomy were performed from 01.01.2018 to 31.12.2019 from which 122 were operated on because of a malignant disease. 51 open and 71 laparoscopic operations were performed. The average age of 37 men and 34 women were 70.59 and 72.14 years, respectively. 50 patients underwent extracorporeal (EA) anastomosis and 21 intracorporeal (IA) anastomosis. Within 30 days the number of complications were 18 in the EA group and 3 in the IA group (p = 0.067). 3 from the EA group and 1 from IA group died within 30 days (p = 0.66). The average length of stay were 9.48 days in the EA group and 6.52 days in the IA group together with the complicated cases (p = 0.001) while 6.35 days and 5.55 days without the complicated cases (p = 0.09). The average duration of operation was 147 minutes in the EA and 146.47 minutes in the IA group (p = 0.11). Conclusion: We found concordance with the literature that there are fewer complications in case of IA which might be related to shorter length of stay. There is no significant difference between the surgical times. Bearing these facts in mind, IA might be suggested for right- sided laparoscopic hemicolectomy.


2020 ◽  
Vol 35 (3) ◽  
Author(s):  
Tayyaba Gul Malik ◽  
Hina Nadeem ◽  
Eiman Ayesha ◽  
Rabail Alam

Objective: To study the effect of short-term use of oral contraceptive pills on intra-ocular pressures of women of childbearing age.   Methods: It was a comparative observational study, conducted at Arif memorial teaching hospital and Allied hospital Faisalabad for a period of six months. Hundred female subjects were divided into two groups of 50 each. Group A, included females, who had been taking oral contraceptive pills (OCP) for more than 6 months and less than 36 months. Group B, included 50 age-matched controls, who had never used OCP. Ophthalmic and systemic history was taken. Careful Slit lamp examination was performed and intraocular pressures (IOP) were measured using Goldman Applanation tonometer. Fundus examination was done to rule out any posterior segment disease. After collection of data, we analyzed and compared the intra ocular pressures between the two groups by using ANOVA in SPSS version 21.   Results: Average duration of using OCP was 14.9 months. There was no significant difference of Cup to Disc ratios between the two groups (p= 0.109). However, significant difference was noted between the IOP of OCP group and controls. (p=0.000). Conclusion: OCP significantly increase IOP even when used for short time period.


Author(s):  
Zeineb Tbini ◽  
Mokhtar Mars ◽  
Mouna Bouaziz

Purpose: The purpose of this study was to investigate T1 relaxation time of the human Achilles tendon, to test its short-term repeatability as well as the minimal detectable change, and to assess the extent that correlate with clinical symptoms. Methods: Twenty asymptomatic volunteers and eighteen patients with clinically and sonographically confirmed tendinopathy were scanned for ankle using a 3 Tesla (T) MR scanner. T1 maps were calculated from a variable flip angle gradient echo Ultra-short echo time sequence (VFA-GE UTE) and inversion recovery spin echo sequence (IR-SE) using a self-developed matlab algorithm in three regions of interest of Achilles Tendon (AT). Signal to Noise Ratio (SNR) between the two sequences was evaluated. INTRA-class Correlation Coefficient (ICC), Coefficient of Variation (CV) and the Least Significant Change (LSC) were calculated, to test short-term repeatability of T1. Subjects were assessed by the VISA-A clinical score. P values less than 0.005 were considered statistically significant. Results: Mean T1 values were 427.09 ± 53.37 ms and 528.70 ± 103.50 ms using IR-SE sequence and 575.43 ± 110.60 ms and 875.81 ± 425.77 ms with VFA-GE UTE sequence in the whole AT for volunteers and patients, respectively. : T1 values showed a significant difference between volunteers and patients (P=0.001). Regional variation of T1 in healthy and tendinopathic AT were greater for VFA-GE UTE sequence than for IR-SE sequence. VFA-GE UTE sequence showed clearly higher SNR compared to IR-SE sequence. Short-term repeatability of T1 values for volunteers showed an LSC of 22% and 14% for IR-SE sequence and VFA-GE UTE sequence, respectively. For patients, LSC was 14% and 5% for IR-SE sequence and VFA-GE UTE sequence, respectively. There was no correlation between T1 and VISA-A clinical score (p>0.005). Conclusion: VFA-GE UTE sequence used for T1 mapping calculation demonstrated short acquisition time and clearly high SNR. Results revealed that T1 relaxation time can be used as a biomarker to differentiate between healthy and pathologic Achilles tendon. However, T1 showed no correlation with the VISA-A clinical score.


2021 ◽  
Vol 22 (3) ◽  
pp. 1417
Author(s):  
Azeem Danish ◽  
Robin Gedschold ◽  
Sonja Hinz ◽  
Anke C. Schiedel ◽  
Dominik Thimm ◽  
...  

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.


2021 ◽  
Vol 28 (Supplement_1) ◽  
Author(s):  
A Abdul Razzack ◽  
D Rocha Castellanos ◽  
A Lopez Mendez ◽  
M Fernando Perez Paz ◽  
S Pothuru ◽  
...  

Abstract Funding Acknowledgements Type of funding sources: None. Background- Patients with small Abdominal Aortic Aneurysms are managed with surveillance as there is currently insufficient evidence to recommend surgical aneurysm repair. Hence, there is a dire need and interest in pharmacotherapy like tetracycline antibiotics to reduce the need for aneurysm repair. Purpose- To determine the efficacy and safety of doxycycline in the management of small abdominal aortic aneurysms. Methods- Electronic databases (PubMed, Scopus, Embase, Cochrane) were searched until 25th November 2020.The primary outcomes were the mean difference (MD) in aneurysm diameter and the odds ratio (OR) calculated to compare the number of individuals referred to Abdominal aortic aneurysm repair in each group. Results- A total of three studies with 572 participants (Doxycycline = 290; Placebo = 282 ) were included in our analysis. Average follow up was a period of 18 months. For AAA expansion, the combined results demonstrated a statistically significant mean difference in expansion rates favoring the placebo groups over the intervention (WMD-0.75, 95%CI 0.12-1.38; p = 0.02;I2 = 0%) There was no statistically significant difference in the efficacy and safety of doxycycline as opposed to placebo groups for referral to AAA surgery (OR 1.01, 95%CI 0.61-1.69; p = 0.96, I2 = 0%) and all-cause mortality(OR 0.51; 95%CI 0.18-1.43; p = 0.20, I2 =0%) Conclusion- Amongst patients with small abdominal aortic aneurysms, doxycycline did not significantly reduce aneurysm growth. Abstract Figure. A) AAA expansion B)Surgery C)Mortality


2002 ◽  
Vol 80 (3) ◽  
pp. 297-304 ◽  
Author(s):  
Mohammed Bajji ◽  
Jean-Marie Kinet ◽  
Stanley Lutts

The effects of salt and osmotic stresses on the germination processes in seeds of the perennial halophyte species Atriplex halimus L. were compared using iso-osmotic concentrations of NaCl and mannitol. The lowest stress intensity delayed germination, while higher doses of NaCl and mannitol reduced final germination percentages. No significant difference occurred between the effects of these solutes on germination percentages or seedling dry weights. At an external osmotic potential of –0.7 MPa, however, the water content of mannitol-treated seedlings was reduced compared to that of seedlings that developed from NaCl-exposed seeds. The K, Mg, and Pi content decreased in seedlings that developed from mannitol-treated seeds while calcium concentration was strongly reduced in those arising from NaCl-treated seeds. Inhibited seeds were able to germinate at levels similar to those of the control after rinsing in deionized water and imbibition in control conditions. Seedlings produced from NaCl pre-treated seeds had a lower Ca and a higher Na content than control seedlings. The effect of salinity on the germination phase of development is mainly due to its osmotic component, and inhibition of germination is reversible. Both salt and osmotic stresses may have an impact on the mobilization of minerals from the seeds to the young seedling, but this effect does not have any consequence on growth processes analysed on a short-term basis.Key words: Atriplex halimus, halophyte, osmotic stress, recovery of seed germination, salinity.


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