Mitochondrial Transfer via Tunneling Nanotubes (TNT) is an Important Mechanism by which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the in vitro and in vivo Models of ARDS

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in pre-clinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the anti-microbial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC anti-microbial effect in the in vivo model of E.coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct co-culture of MSC with monocyte-derived macrophages (MDMs) enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through TNT-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the anti-microbial effect of MSC in vivo.

Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the anti-microbial effect of MSC in ARDS.

Evaluation of the in vitro biological activities of extracts from carob tree and mediterranean oaks

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2009

The in vitro assessment of the synergistic effects of antibiotics and wound dressings on biofilms from diabetic foot pathogens

The impact of biofilm in the effective control of wound microbiome is an ongoing dilemma which has seen the use of different treatment strategies. The effects of wound dressings and antibiotics on both planktonic bacteria and biofilms have been separately evaluated in previous studies. In this current study, the combined antimicrobial effects of some selected wound dressings (silver-impregnated: Acticoat and Silvercel; and honey-impregnated: Medihoney™ Apinate) and antibiotics (ceftazdime and levofloxacin) on Klebsiella pneumoniae and Proteus mirabilis in their quasi-biofilm state were assessed using zone of inhibition (ZOI) test. Before the addition of the wound dressings, bacterial suspension of 108 colony forming units per mL and different concentrations of ceftazidime and levofloxacin (256, 512, 1024 and 5120µg/mL) of a final volume of 1mL were inoculated on Mueller Hinton agar and allowed to dry. Wound dressings cut into circular shapes (2cm diameter) were aseptically placed on the agar plates and incubated at 35 – 37°C for 24 hours. ZOIs associated with Acticoat, Silvercel and Medihoney™ Apinate dressings were compared with that of Atrauman (non-medicated control) dressing. All three dressings showed significant (p < 0.05) biofilm-inhibiting activity against both bacteria at antibiotic concentrations of 1024 and 5120µg/mL with ZOI between 17.5 and 35mm.

Behavioral and synaptic circuit analysis in models of neuropsychiatric disorders: Dissecting the in vivo role of the postsynaptic density proteins nArgBP2 and Shank3 using genetic engineered mice

Dissertation presented to obtain the Ph.D degree in Biology

A business model for an entertainment solution to the in-vehicle infotainment industry

The evolution of mobile technologies that make its presence something ubiquitous and the idea of internet connectivity in every device, often called as the Internet of Things, are pushing a disruption in other industry: the in-vehicle infotainment (IVI). Many companies are trying to enter this new industry that comprises information (weather, news, location services) and entertainment solutions in just one. For that purpose, company X developed a new entertainment solution and intends to bring it to market. This Work Project focuses on creating a business model and an entry mode for the company.

Transport of methotrexate by the in vitro isolated rabbit proximal tubule.

The tubular transport of [3H]methotrexate was studied in isolated nonperfused and perfused superficial proximal tubular segments of rabbit kidneys. Reabsorption represented only 5% of perfused methotrexate, and appeared to be mostly of passive nature inasmuch as it was not modified by reducing the temperature or by ouabain. Cellular accumulation in nonperfused segments and secretion in perfused tubules were highest in the S2 segment and lower in the S3 and S1 segments. Secretion against a bath-to-lumen concentration gradient was observed only in S2 segments (with a maximum methotrexate secretory rate of 478 +/- 48 fmol/mm.min and an apparent Km of transport of 363 +/- 32 microM), and was inhibited by probenecid and folate. The low capacity for methotrexate secretion may be explained by a low capacity of transport across the basolateral membrane of the proximal cell as methotrexate was accumulated only to a low extent in nonperfused tubules (tissue water to medium concentration ratio of 8.2 +/- 1 in S2 segments). During secretion a small amount of methotrexate was metabolized; the nature of the metabolite(s) remains to be defined.

Assessment of the in vitro synergy of daptomycin plus linezolid against multidrug-resistant enterococci

The widespread incidence of enterococci resistant to ampicillin, vancomycin and aminoglycosides, the first-line anti-enterococcal antibiotics, has made the treatment of severe enterococcal infections difficult and alternatives should be explored. We investigated the activity of daptomycin combined with linezolid against three Enterococcus faecalis and four Enterococcus faecium strains resistant to standard drugs used for therapy. Minimum inhibitory concentrations (MICs) were determined by the broth dilution method. Drug interactions were assessed by the checkerboard and time-kill methods. Synergy was defined by a fractional inhibitory concentration index (FICI) of ≤0.5 or a ≥2 log10 CFU/mL killing at 24 h with the combination in comparison with killing by the most active single agent. Indifference was defined by a FICI > 0.5-4.0 or a 1-2 log10 CFU/mL killing compared with the most active single agent. MICs of daptomycin were 2-4 μg/mL for E. faecalis and 2-8 μg/mL for E. faecium. MICs of linezolid were 1-2 μg/mL for all bacteria. In the checkerboard assay, five isolates showed synergism (FICI < 0.5) and two showed indifference (FICIs of 0.53 and 2). Killing studies revealed synergy of daptomycin plus linezolid against four isolates (2.2-3.7 log10 CFU/mL kill) and indifference (1.1-1.6 log10 CFU/mL kill) for the other three strains. Antagonism was not observed. In conclusion, the combination of daptomycin and linezolid had a synergistic or indifferent effect against multidrug-resistant enterococci. Additional studies are needed to explore the potential of this combination for severe enterococcal infections when first-line antibiotic combinations cannot be used.

The in vivo characterization of the DNA repair gene apn-1 in the model organism Caenorhabditis elegans

Les sites apuriniques/apyrimidinique (AP) représentent une forme de dommage à l’ADN hautement mutagène et ce type de dommage peut survenir spontanément ou être induit par une variété d’agents. Afin de préserver la stabilité génomique, deux familles d’endonucléases de type AP, endo-IV et exo-III, sont nécessaires pour contrecarrer les effets mutagènes des sites AP. Malgré l’identification de membres des deux familles dans plusieurs organismes unicellulaire tels que E.coli et S. cerevisiae, aucun membre de la famille endo-IV n’a été identifié chez les organismes multicellulaires à l’exception de C. elegans et de C. briggsae. Nous avons donc décidé d’investiguer l’importance biologique de APN-1 chez C. elegans par l’utilisation d’une approche de knockdown du gène. Dans notre étude, nous avons montré que le knockdown du gène apn-1 chez C. elegans, en utilisant des ARN d’interférence (ARNi), cause une accumulation de mutations spontanées et induites par des drogues résultant en un délai de l’éclosion des œufs ainsi que par une diminution de la survie et de la longévité des vers adultes. De plus, nous avons montré que cette accumulation de mutations mène à un délai dans la progression du cycle cellulaire durant l’embryogénèse, représentant possiblement une explication du délai dans l’éclosion des œufs. Nous avons montré qu’il y avait une augmentation du niveau de mutations dans la gorge des vers, sans toutefois pouvoir confirmer la distribution de APN-1 qui possède une étiquette GFP. Les animaux transgéniques APN-1-GFP n’exprimaient pas suffisamment de la protéine de fusion pour permettre une visualisation à l’aide d’un microscope à fluorescence, mais la protéine a été détectée par immunobuvardage de type western. Les animaux transgéniques APN-1-GFP étaient instables et avaient des phénotypes concordants avec les défauts génétiques. En conclusion, il semble que C. elegans aie évolué afin de retenir un niveau de base de APN-1 jouant ainsi un rôle versatile afin de maintenir l’intégrité génétique d’autant plus que cet organisme semble manquer plusieurs enzymes de la voie de réparation par excision de base.

What is the Web? The Web Architecture

The Introductory Lecture is a discussion about "What is the Web". It involves lots of calling out TLAs and writing them on the blackboard, dividing things into servers, clients, protocols, formats, and the punchline is that the one unique and novel thing about the web is the hypertext link. This follows naturally into the Web architecture - the answer to the question "what is the web".

Senescence-associated β-galactosidase activity in the in vitro ovarian stromal fibroblasts

Introduction: A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, with malignant transformation. Given the high incidence of ovarian cancer and its main origin from the ovarian surface epithelium, as well as the possibility that an epithelial-mesenchymal transition occurs, we evaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6, enzyme whose expression is considered as a marker of replicative senescence. Methods: 48 samples of ovarian cortical fibroblasts from donors without a history of cancer were serially cultured until the end of their replicative life. β-galactosidase activity at pH 6 was quantified in each passage by the chemiluminiscent method. As control, we used ovarian epithelial cell cultures from the same donors. The enzyme activity was also evaluated in fibroblasts previously induced to senescence by exposure to hydrogen peroxide. Results: The analysis of the enzyme activity and the replicative capacity taken together showed that the fibroblast cultures reached the senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions: The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to the epithelialmesenchymal transition that has been proposed to explain their behavior in the genesis of cancer arising from ovarian surface epithelium. Low β-galactosidase activity values at pH 6 would suggest possible inactivation of the response pathways to oxidative stress.

The other in me: the ''in-between" identities of two immigrant autobiographers

This paper looks at how two immigrant autobiographies can be read and understood from a postcolonial perspective. Emphasis is placed on the theoretical framework of Postcolonial Studies and how the presence of a hegemonic “other” can influence the formation of identity. More specifically, I will show how the culture of the “colonizer” is internalized and how the two authors assimilate the cultural dominant before immigrating.

Influence of exogenous fibrolytic enzyme level and incubation pH on the in vitro ruminal fermentation of alfalfa stems

A series of in vitro experiments was carried out to examine the impact of enzyme application rate and incubation medium pH on the rate and extent of fermentation of alfalfa stems. In Experiment 1, a commercial enzyme product (Liquicell 2500, Specialty Enzyme and Biochemicals, Fresno, CA, USA) was added to alfalfa stems at six levels: 0, 0.51, 1.02, 2.55, 5.1, and 25.5 mu l/g (control and L1-L5, respectively) to forage DM in a completely randomized design, with a factorial arrangement of treatments. Rate and extent of fermentation and apparent organic matter degradation (OMD) were determined in vitro, using a gas production technique. Addition of enzyme linearly increased (P < 0.01) gas production for up to 12 h (68.9, 70.9, 67.6, 67.9, 71.9, and 74.9 ml/g OM for control, L1-L5, respectively) and OMD for up to 19 h incubation (0.425, 0.444, 0.433, 0.446, 0.443, and 0.451 for control, L1-L5, respectively), but no increases (P > 0.05) were detected thereafter. In Experiment 2, the effect of the same enzyme as used previously (added at 0.51 mu l/g forage DM, directly into the incubation medium), and buffer pH were examined using the ANKOM system, in a completely randomized design. Incubation medium pH was altered using 1 M citric acid, in order to obtain target initial pH values of 6.8 (control, no citric acid added), 6.2, 5.8, and 5.4. Actual initial pH values achieved were 6.72, 6.50, 6.20, and 5.72. Lowering the pH decreased (P < 0.01) dry matter disappearance (DMD) at 18 h incubation (0.339, 0.341, 0.314, and 0.291 for 6.72, 6.50, 6.20, and 5.72, respectively), whereas enzyme addition increased (P < 0.05) DMD at 24 h (0.363 versus 0.387 for control and enzyme-treated, respectively). Addition of enzyme increased (P < 0.05) neutral detergent fibre (NDF), acid detergent fibre (ADF), and hemicellulose (HC) degradation at pH 6.50 (0.077 versus 0.117; 0.020 versus 0.051; 0.217 versus 0.270 for control and enzyme-treated NDF, ADF and hemicellulose degradation, respectively) and 6.72 (0.091 versus 0.134; 0.041 versus 0.079; 0.205 versus 0.261 for control and enzyme-treated NDF, ADF and HC degradation, respectively). It is concluded that the positive effects of this enzyme product were independent of the pre-treatment period, but pH influenced the responses to enzyme supplementation. Under the conditions of this experiment, exogenous fibrolytic enzymes seemed to work better at close to neutrality ruminal pH conditions. (C) 2006 Elsevier B.V. All rights reserved.

Technical note: A proposed method to determine the extent of degradation of a feed in the rumen from the degradation profile obtained with the in vitro gas production technique using feces as the inoculum

A method is proposed to determine the extent of degradation in the rumen involving a two-stage mathematical modeling process. In the first stage, a statistical model shifts (or maps) the gas accumulation profile obtained using a fecal inoculum to a ruminal gas profile. Then, a kinetic model determines the extent of degradation in the rumen from the shifted profile. The kinetic model is presented as a generalized mathematical function, allowing any one of a number of alternative equation forms to be selected. This method might allow the gas production technique to become an approach for determining extent of degradation in the rumen, decreasing the need for surgically modified animals while still maintaining the link with the animal. Further research is needed before the proposed methodology can be used as a standard method across a range of feeds.

Alternative methodologies - stretching the in vitro box

Current gas-based in vitro evaluation systems are extremely powerful research techniques. However they have the potential to generate a great deal more than simple fermentation dynamics. Details from four experiments are presented in which adaptation, and novel application, of an in vitro system allowed widely differing objectives to be examined. In the first two studies, complement methodologies were utilised. In such assays, an activity or outcome is inferred through the occurrence of a secondary event rather than by direct observation. Using an N-deficient incubation medium, the increase in starch fermentation, when supplemented with individual amino acids (i.e., known level of N) relative to that of urea (i.e., known quantity and N availability), provided an estimate of their microbial utilisation. Due to the low level of response observed with some arnino acids (notably methionine and lysine), it was concluded, that they may not need to be offered in a rumen-inert form to escape rumen microbial degradation. In another experiment, the extent to which degradation of plant cell wall components was inhibited by lipid supplementation was evaluated using fermentation gas release profiles of washed hay. The different responses due to lipid source and level of inclusion suggested that the degree of rumen protection required to ameliorate this depression was supplement dependent. That in vitro inocula differ in their microbial composition is of little interest per se, as long as the outcome is the same (i.e., that similar substrates are degraded at comparable rates and end-product release is equivalent). However where a microbial population is deficient in a particular activity, increasing the level of inoculation will have no benefit. Estimates of hydrolytic activity were obtained by examining fermentation kinetics of specific substrates. A number of studies identified a fundamental difference between rumen fluid and faecal inocula, with the latter having a lower fibrolytic activity, which could not be completely attributed to microbial numbers. The majority of forage maize is offered as an ensiled feed, however most of the information on which decisions such as choice of variety, crop management and harvesting date are made is based on fresh crop measurements. As such, an attempt was made to estimate ensiled maize quality from an in vitro analysis of the fresh crop. Fermentation profiles and chemical analysis confirmed changes in crop composition over the growing season, and loss of labile carbohydrates during ensiling. In addition, examination of degradation residues allowed metabolizable energy (ME) contents to be estimated. Due to difficulties associated with starch analysis, the observation that this parameter could be predicted by difference (together with an assumed degradability), allowed an estimate of ensiled maize ME to be developed from fresh material. In addition, the contribution of the main carbohydrates towards ME showed the importance of delaying harvest until maximum starch content has been achieved. (c) 2005 Elsevier B.V. All rights reserved.