Post Incubation Research Articles

Modulation of Apoptosis and Autophagy by Melatonin in Juglone-Exposed Bovine Oocytes.

Melatonin, an antioxidant hormone secreted by the pineal gland, has been recognized as a regulator for numerous biological events. The deleterious effects of juglone, a polyphenolic extract of walnut trees, on embryo development has been previously reported. In the current study, we aimed to display the impact of melatonin administrated during in vitro oocyte maturation (IVM) on juglone-treated oocytes. Thus, in vitro matured oocytes were collected after 24 h post incubation with juglone in the presence or absence of melatonin. Reactive oxygen species (ROS), glutathione (GSH) content, mitochondrial distribution, and the relative abundance of mRNA transcription levels were assessed in oocytes, in addition, oocytes were in vitro fertilized to check the competency levels of oocytes to generate embryos. We found that administration of melatonin during the maturation of oocytes under juglone stress significantly improved the cleavage rate, 8-16 cell-stage embryos and day-8 blastocysts when compared to the sole juglone treatment. In addition, the fluorescence intensity of ROS increased, whereas the GSH decreased in juglone-treated oocytes compared to melatonin-juglone co-treated and untreated ones. Additionally, a significant increase in the mitochondrial aberrant pattern, the pattern that was normalized following melatonin supplementation, was observed following juglone administration. The mRNA analysis using RT-qPCR revealed a significant upregulation of autophagy and oxidative-stress-specific markers in the juglone-treated group compared to the co-treatment and control. In conclusion, the study reveals, for the first time, a protective effect of melatonin against the oxidative stress initiated following juglone treatment during the in vitro maturation of oocytes.

Piperazine-modified dendrimer achieves efficient intracellular protein delivery via caveolar endocytosis bypassing the endo-lysosomal pathway.

Intracellular protein delivery has been a major challenge due to various physiological barriers including low proteolytic stability and poor membrane permeability of the biologics. Nanoparticles were widely proposed to deliver cargo proteins into cells by endocytosis, however, the materials and complexes with proteins are often entrapped in endosomes and subject to lysosome degradation. In this study, we report a piperazine modified dendrimer for stabilizing the complexes via a combination of electrostatic interaction and hydrophobic interactions. The complexes show rapid cell internalization and the loaded proteins are released into the cytosols as early as half an hour post incubation. Mechanism study suggests that the complexes are endocytosed into cells via caveolae-based pathways, which could be inhibited by inhibitors such as genistein, filipin III, brefeldin A and nystatin. The phenylpiperazine-modified polymer enables the delivery of cargo proteins with reserved bioactivity and show high permeability in three-dimensional cell spheroids. The results prove the beneficial roles of phenylpiperazine ligands in polymer-mediated cytosolic protein delivery systems. STATEMENT OF SIGNIFICANCE: We synthesized a list of piperazine and derivatives modified dendrimers as cytosolic protein delivery vectors via facile reactions. Phenylpiperazine modification enables the efficient protein binding through the combination of electrostatic, hydrogen bonding and hydrophobic interactions. Phenylpiperazine modified dendrimers were internalized into the cells via a caveolae-based endo/lysosome-independent path and could release the cargo proteins into the cytosols as early as half an hour post incubation. Phenylpiperazine modified dendrimers delivered cargo proteins with reserved bioactivity and showed high permeability in three-dimensional cell spheroids.

Natamycin as a safe food additive to control postharvest green mould and sour rot in citrus.

The purpose of this study was to explore the potential inhibitory mechanism and assess the feasibility of natamycin as an antifungal agent in the utilization of citrus storage. In this study, the mycelial growth, spore germination as well as germ tube elongations of Geotrichum citri-aurantii and Penicillium digitatum were significantly inhibited by natamycin treatment. The relative conductivities of G. citri-aurantii and P. digitatum mycelia were increased as time went by and the damages of plasma membranes were up to 17.43% and 28.61%. The mitochondria abnormalities and vacuolation were also observed in the TEM. Moreover, the sour rot and green mould decay incidences were reduced to 18.33% and 10% post incubation with G. citri-aurantii and P. digitatum under 300 mg L-1 natamycin application, respectively. For the citrus storage experiment, there was no significant difference in edible rate, juice yield, total soluble solid (TSS) content, titratable acid (TA) and decay incidences of the 'Newhall' navel orange fruit treated with 300 mg L-1 natamycin stored for 90 d. Natamycin could decrease the expansions of green mould and sour rot and maintain quality and improve storability on citrus fruit. This work explores the potential inhibition mechanism of natamycin G. citri-aurantii and P. digitatum and assesses the feasibility of natamycin as an antifungal agent in the utilization of citrus storage.

The role of human microglia and microglial LRRK2 in tau pathogenesis

AbstractBackgroundThe G2019S mutation in leucine‐rich repeat kinase 2 (LRRK2) is the most common genetic determinant of Parkinson’s disease (PD), reported in both familial and sporadic PD cases. Even though tau‐positive pathology features prominently in the G2019S PD patients, the role of LRRK2 in tau pathogenesis remains poorly understood. Human microglia – the chief brain‐resident phagocytes of the innate immune system ‐ become one of the highest LRR2K expressing cells upon IFN‐y stimulation. Moreover, microglia have been recently implicated in increasing pathological tau transmission by secreting exosome‐bound, seeding‐competent tau into the extracellular space. Using human iPS‐macrophages, a simple but physiologically relevant and genetically tractable model of human microglia, we set out to define the role of microglial LRRK2 in uptake, degradation, and secretion of disease‐associated tau protein.MethodWe have developed a quantitative phagolysosomal proteolysis assay based on dequenching of DQ‐BSA fluorogenic substrate attached to silica microspheres, allowing us to investigate cell‐autonomous phagocytic and degradative capacity of G2019S, LRRK2 KO and isogenic control iPS‐macrophages. In parallel, we have optimised purification and in vitro aggregation of endotoxin‐free, human recombinant 2N4R tau to study tau‐induced phagolysosomal proteolysis deficits in the said iPS‐macrophages.ResultWe have found that human iPS‐macrophages readily internalize extracellular tau monomer and fibrils, and LRRK2 is required for macrophage uptake of tau monomer. Degradation of internalized tau is concentration and incubation‐length dependent regardless of genotype. However, G2019S macrophages show deficits in degradation of internalized tau fibrils. Moreover, non‐degraded tau fibrils present within G2019S cell‐ lysates 24h post incubation retain seeding competency and induce aggregation of naïve tau monomer in K11 tau RT‐QuIC assay, unlike tau fibrils internalized by LRRK2 KO macrophages.ConclusionLRRK2 expressed by iPS‐macrophages is important for monomeric tau entry, degradation, and seeding capacity of internalized tau fibrils. Future studies will focus on elucidating the molecular mechanisms behind the LRRK2 and tau interaction.

Early-Stage vs. Growth-Stage Startups: Examining the Differences in the Perception of Factors Instrumental to Success

A stream of research available in the literature focuses on startup success factors, although there is a lack of consensus in determining what these factors are. In general, there are two critical groups of success factors, organizational and entrepreneurial, both considered to be integral parts of internal startup environment. Since the perception of early-stage (incubation stage) and growth stage (post incubation stage) startup entrepreneurs differ significantly they tend to perceive important success factors distinctively. As a result, attributes of success differ considerably. This paper aims at capturing internal startup environment as it is considered to be crucial in shaping startup’s success in contrast to external environment that is more remote and (usually) not manageable.
 Primary data were collected using an online survey questionnaire and quantitative approach was used; data were analysed using descriptive statistics and Mann-Whitney U test. The final research sample consisted of 70 startups and the findings indicate that the perception of internal environment and its contribution to overall success differs significantly from the perspective of early-stage and growth stage startup entrepreneurs. Out of analysed internal factors, statistically significant differences were identified for prior experience in starting the company and self-confidence (individual’s/entrepreneur’s category factors) and internal startup organization and team-level competencies (organizational category factors).
 The contribution of this research is evident in identifying important internal factors that determine startup’s success (that aspiring startup founders can benefit from) and providing an insight into the startup community in Croatia, thus contributing to a scarce empirical literature on the topic available. Limitations of the research include sample size and subjective assessment of the success factors, so recommendations for future research include repeating the research on a larger sample, covering the international level and widening a set of key-performance indicators that can be assessed at a higher level of rigour with objective metrics.

PSV-A-12 The Influence of Calcium Dose and Olive Meal on in-Vitro Rumen Fermentation Characteristics

Abstract Rumen fluid from three beef steers (480 ± 10 kg), fitted with rumen canulae, were used to investigate the impact of Ca dose and olive meal on in vitro rumen fermentation characteristics. Steers were fed a high concentrate finishing diet for 21d, and rumen fluid was collected from each steer 2h post-feeding. A 2 x 4 factorial arrangement of treatments was used for this experiment. Factors included: 1) 0 or 5% olive meal and 2) Ca dose: 0, 0.02, 0.04, and 0.08% Ca from CaCl2. A McDougall’s buffer-rumen fluid mixture (1:1; 30 mL) was added to conical tubes containing 0.5g of the ground basal diet and incubated at 39°C for 0, 4, 8, and 12h (5 replicates per treatment per time point). After incubation, supernatant was removed for VFA analysis and the remaining digesta was dried to determine DM disappearance (DMD). At 4 and 8h post incubation digestion tubes containing 0.04% Ca had greater (P < 0.001) DMD when compared to all other Ca doses. At 12h post incubation, DMD was greater (P < 0.001) in digestion tubes containing 0.02% and 0.08% Ca compared to all other Ca doses. At 8h post incubation, molar proportions of acetic acid were greater (P < 0.03) in digestion tubes containing olive meal compared to no olive meal and were greater (P < 0.001) in digestion tubes containing 0.08% Ca compared to all other Ca doses. At 12h post incubation, isobutyric acid (P < 0.01) and butyric acid (P < 0.02) were greater in digestion tubes containing 0.02% and 0.04% Ca compared to all other Ca doses. Butyric acid was lesser (P < 0.02) with olive meal inclusion at 12h. Total VFA concentrations were similar across treatments. These data suggest that Ca and olive meal can impact in vitro fermentation characteristics.

Effect of aging on monocyte phagocytic and inflammatory functions, and on the ex vivo inflammatory responses to lipopolysaccharide, in horses

Studies investigating age-related changes in the function of monocytes are currently limited for horses. Thus, the main goal of this study was to determine the effect of aging on monocyte phagocytic capacity and pro-inflammatory cytokine responses to bacterial lipopolysaccharide (LPS). A second goal of this work was to examine the effect of aging on the inflammatory cytokine responses to LPS in a whole blood ex vivo model. Seven healthy young adult (4–6 years of age) and seven healthy senior horses (>20 years of age) were enrolled. Phagocytosis of E. coli, and pro-inflammatory cytokine (TNFα) responses to LPS, were measured in monocytes by flow cytometry. Gene expressions of pro-inflammatory cytokines (TNFα, IL-1β, IL-6, IL-8, IL-18, CCL-5, CCL-2) were measured in peripheral blood mononuclear cells (PBMCs) and whole blood by RT-qPCR post incubation for 2 h or 6 h with a low (0.01 µg/mL) or a high (1 µg/mL) dose of LPS. Two sets of statistical models were applied to compare the age groups, one adjusted, and one unadjusted for the horses’ body condition scores (BCS). The percentage of monocytes that phagocytosed E. coli after 2 h of incubation was significantly lower in senior compared to young adult horses in the BCS-adjusted model. In the senior group, the expression of IL-1β in 2 h-0.01 µg/mL LPS-stimulated PBMCs was significantly higher than in the young adult group (BCS-adjusted and unadjusted models). In senior horses, expressions of IL-8 and IL-6 in whole blood samples stimulated for 6 h with 0.01 µg/mL LPS and for 2 h with 1 µg/mL LPS, respectively, were significantly lower than in young adult horses (BCS-adjusted models). The results of this study suggest that the phagocytic function of monocytes, as well as their IL-1β response to LPS may be altered in senior horses. In addition, the whole blood IL-8 and IL-6 gene expression responses to LPS may be insufficient in senior horses. While investigation of the effect of BCS on monocyte functions and whole blood pro-inflammatory LPS-responses was not a major goal of this work, it appears that adiposity may play a role in innate immune cell function, as significant differences between the age groups were often not apparent until the models were adjusted for BCS.

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

PB1857: NRF2 MODULATORS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: A POSSIBLE NEW THERAPEUTIC APPROACH

Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of resting clonal B lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. This disease has a notable heterogeneous clinical course, with different prognosis and distinct treatment needs. Oxidative stress (OS) is one of the mechanisms important in CLL development and progression. OS state consists in an unbalance between the production of reactive oxygen species (ROS) and the ability to neutralize them by antioxidant defenses. The nuclear factor erythroid 2–related factor 2, NRF2, is a transcription factor that regulates the expression of several antioxidants and detoxification systems and works as a protective mechanism in normal cells against malignant transformation. However, during the tumorigenic process, NRF2 protects tumor cells from OS effects compromising the therapeutic efficacy. Aims: The study aims to evaluate the therapeutic potential of two NRF2 modulators, Brusatol and ML385, in B cells from CLL patients. Methods: Peripheral blood mononuclear cells (PBMCs) from 13 CLL patients (stage initial, Binet A/B, n=8; advanced stage, Binet C, n=5) were isolated by density gradient using Ficoll-Paque Premium. PBMCs where immediately incubated in absence and presence of Brusatol and ML385. The cytotoxic effect was evaluated 24h and 48h after incubation by fluorometric microculture cytotoxicity assay. After 24h incubation with Brusatol (100nM) and ML385 (100μM) cells were stained with anti-CD5-PerCP, anti-CD19-APC and Annexin V, to assess cell death by flow cytometry. The statistical analysis was carried out in the total CLL patient group and in risk groups. The results were considered statistically significant when p<0.05. Results: CLL patients presented 93,3% (±3.7%) of neoplastic B cells (CD5+/CD19+), 3,7% (±3,0%) of normal B cells (CD5-/CD19+) and 2,7% (±1,2%) of normal T cells (CD5+/CD19-), with no statistical differences regarding staging groups. Brusatol induced a decreased survival rate in CLL cells comparing to ML385, being this effect dependent on the dose and disease stage. Brusatol induced a higher reduction of cell viability in PBMCs from advanced disease patients (Binet C). ML385 did not induced a significant decrease in cell survival, nevertheless its effect is more marked in advanced stage patients at 48h post incubation. IC50 was only achieved at 48h in advanced stage patients and corresponds to 65nM for Brusatol and to 114µM for ML385. NRF2 modulators induced a cytotoxic effect mainly mediated by apoptosis. Cytotoxicity induced by both drugs was more evident in neoplastic B cells, with no significant cytotoxicity in normal B and T cells. In PBMCs incubated with Brusatol an increase of neoplastic B cells in apoptosis about 68% was observed (Brusatol: 91,9% ± 7,1%; Control: 23,7% ± 4,3%), and of 34% in cells incubated with ML385 (ML385: 57,9% ± 10,2%; Control: 23,7% ± 4,3%). No differences of apoptotic cells were observed in advanced stage groups. Summary/Conclusion: Our results showed that neoplastic cells from CLL patients were sensitive to NRF2 modulators, suggesting that these drugs may constitute a new therapeutic approach in CLL patients, particularly in CLL patients in advanced stage. We also demonstrated the low cytotoxic effect of these modulators in normal lymphocytes and its higher efficacy in neoplastic cells from advanced stage patients. Altogether, these results show that these NRF2 modulators have an effect directed to the neoplastic cell and suggest a possible involvement of NRF2 on CLL development and progression. # equally contributing authors.

In vitro, effects of Praziquantel and alcoholic and aqueous crude extracts of Citrullus colocynthis and Eucalyptus globulus on the viability of protoscoleces of Echinococcus granulosus

Iraqi flora is rich in plants and the possibility of finding new antimicrobial agents still widely ahead, therefore, the present work evaluated the effects of alcoholic and aqueous crude extracts of Citrullus colocynthis; Eucalyptus globulus leaves and Praziquantel drug (as scolicidal agents) on the viability of Echinococcus granulosus protoscoleces in vitro. Live protoscoleces obtained from the liver of naturally infected sheep. The viability of protoscoleces was determined by their motility and flame cells activity and staining method using 0.1% eosin solution. The number of viable protoscoleces was counted and distributed into test tubes (4 tubes for each treatment of different concentrations) and 2 tubes were kept as control without treatments. The percentage viability of protoscoleces was ranged between 93.7 to 95.3. Praziquantel drug reduced the survival of protoscoleces during seven sequenced days post incubation. Drug effects showed more influence in both concentrations of 450 μg/ml from day 4 and 600 μg/ml from day 3 post incubation, respectively. The methanolic extract of C. colocynthis reduced the viability of protoscoleces during eight sequenced days post incubation from 96% to zero%. The effects were marked highly in the concentrations of 300 μg/ml from day 3 post incubation. Aqueous fruit extracted decreased in the viability of protoscoleces. In the minimum used concentration (75μg/ml), the viability of protoscoleces reduced from 96% to reach zero% at day eight post incubation, whereas in the maximum concentration used (300 μg/ml), the viability reached to zero% after four days. All concentrations of leaves methanolic and aqueous extracts of E. globulus have an antiparasitic activity against the protoscoleces viability, but the greater effect was to 600 μg/ml that kill all protoscoleces in 6 days after treatment.

Evaluation of the In Vitro Cytotoxicity of Silver Nanoparticles on PBMC Cells Using MTT Assay

Background: Silver Nanoparticles are extensively studied by the scientific community for therapeutic applications. With respect to the fundamental pillars of bioethics “Primum non nocere” equal emphasis should be given to evaluate the toxicological perspectives of Silver nanoparticles. This study aims at evaluating the InVitro cytotoxic effects of Silver nanoparticles synthesized using hesperidin. Aim: To study the In Vitro cytotoxicity of silver nanoparticles on PBMC cells using (3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Methods: Synthesized silver nanoparticles at various concentrations are incubated with peripheral blood mononuclear cells (PBMC). After 24 hours MTT is added to the mixture to evaluate the cell viability post incubation. Yellow MTT (a tetrazole) which is reduced to purple formazan in the mitochondria of living cells. The absorbance of this colored solution can be quantified by measuring at 570 nm by a spectrophotometer. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells. Results: ?.Conclusion: Silver Nanoparticles do not exhibit any significant cytotoxicity on PBMCs and also there were no dose dependent trends in the results.

Cytotoxic Effect of Red Sandal Mediated Silver Nanoparticles Mouthwash Using Brine Shrimp Lethality Assay: An In-vitro Study

Background: Silver Nanoparticles (AgNP) plays a crucial role in nanoscience, nanotechnology and nanomedicine. AgNP has antibacterial, antifungal, antiviral, anti-inflammatory, anti-antigenic, anticancer and cytotoxic properties. Cytotoxic effects of mouthwashes on the gingival tissue have always been a priority. Pterocarpus santalinus, with the common name of red sandalwood. The aim of the study is to evaluate the cytotoxic effect of red sandal mediated silver nanoparticles mouthwash using brine shrimp lethality assay.&#x0D; Materials and Methods: The mouthwash was prepared using red sandal mediated silver nanoparticles, ethanol, distilled water, sucrose, sodium benzoate, clove oil, sodium dodecyl phosphate with 0.1% in concentration. The methodology includes green synthesis of Red sanders mediated Silver Nanoparticles synthesis followed by tests for Cytotoxic effect using mouthwash.&#x0D; Results: Red sanders mediated silver nanoparticles have high potent cytotoxic potential, it has been proved with the help of brine shrimps. LD50 concentration was obtained to be 80µL, with half the population of nauplii in the respective well surviving post incubation. &#x0D; Conclusion: From the observed result, it has been concluded that Pterocarpus santalinus/red sandal mediated silver nanoparticles mouthwash has a considerably moderate cytotoxic effect. It is a rich source of various potential phytochemicals and is a simple, rapid, stable and cost effective method for nano based medical applications.

Influence of combined yeast culture and enzymatically hydrolyzed yeast on in vitro ruminal fermentation in contrasting feed substrates.

Feed additives such as live yeast cultures have increasingly been used in ruminant feeds to improve animal performance and feeding efficiency. However, it is not clear how inactive combined yeast cultures affect ruminal gas production, fermentation kinetics and efficiency. Therefore, this study was done to determine the influence of incubating different substrates with a combined yeast culture + enzymatically hydrolyzed yeast (YC + EHY) on in vitro ruminal gas production, fermentation kinetics and metabolizable energy. Six contrasting substrates (Trichantera gigantea and Glircidia sepium leaves, Brachiaria hybrid (cv. Mulato II) leaf + stem and leaf only, Cynodon nlemfuensis and a commercial concentrate dairy feed) were incubated with and without YC + EHY in buffered rumen fluid and gas production measured at 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48 and 72 h post incubation. In vitro fermentation parameters (a, b, a + b and c) were unaffected by YC + EHY except for the lag phase in T. gigantea, which that reduced by 31.3% when it was incubated with YC + EHY. Supplementation with YC + EHY also did not affect metabolizable energy, 72 h organic matter digestibility, 24 h gas or CH4 production within substrate. However, cumulative gas and methane production at peak fermentation in the commercial concentrate feed was reduced by 20% when incubated with YC + EHY. It was concluded that YC + EHY has the potential to improve microbial colonization of T. gigantean substrates and reduce gas and methane production at peak fermentation in commercial concentrate feeds. © 2021 Society of Chemical Industry.

Effect of water deficits on the growth and secondary metabolites of Salvia dolomitica Codd. and Salvia namaensis Schinz

Background: Salvia dolomitica Codd. and Salvia namaensis Schinz. are indigenous to southern Africa and are used as medicinal plants in folk medicine.Aim: This study aimed to assess the effects of different levels of water deficit treatments on the growth, concentration of secondary metabolites, and anti-Fusarium oxysporum activity of S. dolomitica and S. namaensis.Setting: Experiments were carried out on the Bellville campus of the Cape Peninsula University of Technology, Cape Town.Methods: Four weeks old seedlings of the two species were subjected to 3-, 6-, 9- and 12-day water deficit regimes. Secondary metabolites such as polyphenols, alkaloids, and flavanols were assessed using spectroscopic methods. The anti-fungal activities of crude extracts obtained from plants were evaluated in a micro-dilution bioassay.Results: In all treatments, the plant height, crown width, number of stems and leaves, and fresh and dry weights reduced with increased water deficits. Acetone extracts from all treatments showed anti-fungal activity. However, extracts from the treatment with moderate water deficit (6-day watering interval) recorded significantly (P 0.01) better inhibition of F. oxysporum at the 18 h post incubation than the commercial fungicide, Mancozeb.Conclusion: This research has revealed that mild to moderate water deficit level favours the accumulation of alkaloids in S. dolomitica. Meanwhile, mild to severe water deficit significantly lowered flavanol content in S. namaensis. There was a correlation between the increase in total alkaloid contents and the enhanced anti-fungal activity of extracts of S. dolomitica. The present findings pave the way for optimised cultivation of medicinal plants and development of bioactive natural products.

PSXVI-22 The influence of Propionibacteria on in vivo rumen fermentation characteristics and in vitro lactic acid clearance rate in fistulated steers fed moderately high concentrate diets

Abstract Six steers fitted with ruminal cannulae were used in a crossover design to examine the impact of direct fed microbial (DFM) supplementation on in vivo rumen fermentation characteristics and in vitro lactic acid clearance. Steers were fed a moderately high concentrate -1∙day-1 of 4.45x 1010 CFU/g of Propionibacteria acidipropionici). Treatments were administered daily, directly into the rumen via the cannula as a single bolus dose at the time of feeding. Rumen contents were sampled two h post-feeding daily and analyzed for short chain fatty acids (SCFA). On d 7 and 14, rumen fluid was collected from all steers and subjected to an in vitro lactic acid clearance challenge. Lactic acid and SCFA concentrations were determined at 3, 6 and 9 h post incubation. After d 14, all cattle received the basal diet for 21 d. On d 22, treatment crossover was implemented and the experiment repeated. Animal was considered the experimental unit and data were analyzed as a crossover design using Proc Mixed in SAS. In vivo propionic acid concentrations were greater (P &amp;lt; 0.05) and total SCFA tended (P &amp;lt; 0.06) to be greater in rumen fluid from steers receiving DFM. D- and total lactic acid concentrations were lesser (P &amp;lt; 0.05) at 3 h post in vitro incubation for steers receiving DFM. D-, L+, and total lactic acid concentrations were similar between treatments at all other timepoints. In vitro propionic acid and total SCFA concentrations were greater (P &amp;lt; 0.05) and acetic acid concentrations lesser (P &amp;lt; 0.05) in steers receiving DFM. These data indicate that the DFM Propionibacteria acidipropionici, alters in vivo and in vitro rumen fermentation characteristics.

In Vitro Modelling of Barrier Impairment Associated with Gastro-Oesophageal Reflux Disease (GERD).

PurposeA novel experimental model based on a 3D reconstructed human oesophageal epithelium model (HO2E) has been developed to investigate the structural and functional changes of the oesophageal epithelium following exposure to a solution of HCl 0.1 N (pH = 1.2) mirroring GERD microenvironment condition.MethodsThe barrier structure modification after the exposure to the acid solution on HO2E tissues was investigated immediately after damage induction and after 1 hour post incubation and compared to HO2E tissues exposed to phosphate buffered saline solution. Immunofluorescence (IF) was applied to quantify the expression and localization of barrier function proteins: Claudin-1 (CLDN-1), Claudin-4 (CLDN-4), Zonulin-1 (ZO-1), E-Cadherin and Mucin-1 (MUC1). Barrier functionality was measured by TEER.ResultsIn the acidic microenvironment, TEER measurement has shown some limitations and results were not applicable, whereas the evaluation of protein localization and quantification provided clear and robust evidence of the damage which occurred to the epithelium barrier structure. CLDN-4 expression significantly decreased after exposure to acid. ZO-1 protein appeared upregulated immediately after exposure to HCl and was mainly localized in the cytoplasm and not on the cell membrane. This different localization was also observed for CLND-1. CLDN-1, MUC1 and, to a lower extent, ZO-1 expression increased during the post-incubation period.ConclusionThe relevant tissue biomarkers identified, CLDN-1 and MUC1, can be used to monitor TJ structure and epithelial barrier recovery after acid-induced damage which, in our experimental conditions, were non-destructive and suitable for recovery studies. The established model can be useful to investigate the mechanism of action of formulations acting on this specific pathophysiological condition and/or designed to potentiate the physiological defense mechanisms of oesophageal mucosa.

Exploring the role of Malaysian research university-based incubators in facilitating the entrepreneurial process

Objective of the study: To clarify the role of university-based incubators in facilitating the entrepreneurial process by elucidating the value-add of the incubation activities through the lens of real options-driven theory and resource-based view theory.Methodology/approach: An in-depth case study of five Malaysian research-led universities is conducted through thirteen semi-structured interviews with UBI managers and staff.Originality/Relevance: Integrating real options-driven theory and resource-based view to understand the roles of UBI in supporting the entrepreneurial process, elaborated in the three stages of incubation: pre-main-post incubation phase.Main results: UBI has a decisive role in facilitating entrepreneurial process through selecting potential entrepreneurs, mobilising the optimal mix of tangible and intangible resources, and monitoring the entrepreneurial process. All the five UBIs displayed interorganisational relationships with the various stakeholders and have capable incubator manager as a linchpin to facilitate the incubation process from pre to main incubation phase. However, there is little evidence on aftercare post incubation services to support the entrepreneurial growth stage, which are crucial for their continuing survival and growth.Theoretical/methodological contributions: It adopts a multi-theoretical lens of real options-driven theory and resource-based view theory to explain the roles of UBIs in the entrepreneurial process.Social/management contributions: It provides insights, best practices, and frameworks regarding the incubation process of Malaysian UBIs, and how its roles can be enhanced to drive more successful entrepreneurial processes.

Molecular characterization, expression, and apoptosis regulation of siva1 in protogynous hermaphrodite fish ricefield eel (Monopterus albus).

Siva1, which induces extensive apoptosis, has been well characterized. To elucidate the molecular function of Siva1 in ricefield eel, molecular characterization and phylogenetic analysis were performed, and the mRNA expression in the ovary at different developmental stages and ovary tissues exposed to H2O2 and Z-VAD-FMK in vitro were also evaluated. The results indicated that ricefield eel Siva1 was highly conserved and contains three conserved motifs, despite 83 amino acid differences upstream of the initiation codon. Phylogenetic analysis demonstrated that ricefield eel Siva1 clusters together with the Siva1 protein of the other fish, with high sequence homology with that of Lates calcarifer. Quantitative real-time polymerase chain reaction analysis showed high siva1 expression levels in the ovary and low expression levels in the liver. The higher mRNA levels of siva1 were detected in the IE and IM, and the lower siva1 mRNA levels were found in the OM, IL, and TE during gonadal development. Additionally, siva1 expression levels in the ovarian tissues were significantly increased at 1h post incubation (hpi) with H2O2 and then significantly decreased at 2 hpi; however, siva1 expression was upregulated significantly at 4 and 8 hpi, similar to the patterns observed with caspase3, which was used as a molecular marker of apoptosis. Moreover, the siva1 mRNAs were elevated significantly than that in control groups at 1 hpi, but the expression of siva1 was down-regulated dramatically at 2, 4, and 8 hpi, which were similar with that of caspase3 expression profiles after Z-VAD-FMK incubation. What's more, Pearson's correlation coefficients showed strongly positive relationships between siva1 and caspase3. These findings suggest that Siva1 plays an important apoptosis role in gonadal development of ricefield eel.

Newly isolated coliphages for bio-controlling multidrug-resistant Escherichia coli strains

This study presents a novel coliphage-based approach for biocontrol of Multidrug-Resistant (MDR) Escherichia coli (E. coli) and coliforms in wastewater and improve the physicochemical water quality . Newly isolated coliphages and E. coli strains were isolated from the Rosetta branch of River Nile and outlet points of five associated drains. Coliphages were characterized by transmission electron microscopy and coat protein-gene analyses. The isolated coliphages PR01, PR02, and PR03 belonged to the Siphoviridae, Myoviridae, and Podoviridae families, respectively. They exhibited, separately or in a cocktail, a wide host range pattern against wild E. coli strains with higher specificity to strains isolated from drains (87%) than those of the Rosetta branch (72%). Phage-sensitive E. coli strains exhibited elevated antibiotics resistance patterns, suggesting that they are MDR strains. Interestingly, the efficacy of phages cocktail (PR01/PR02/PR03) controlled the growth of wild MDR E. coli strains and coliform populations under laboratories conditions. The kinetic trend in removal efficiency (%) for coliforms was gradually increasing in a time-dependent manner and peaked at 12 h post incubation with phages mixture. In all water samples, total and fecal coliforms revealed at least a 30-fold reduction post 12 h of treatment with a mixture of coliphages. Physicochemical analyses of phage-treated wastewater showed reducing levels in biological oxygen demand (BOD), chemical oxygen demand (COD), turbidity, electric conductivity (EC), ammonia (NH3), total dissolved solids (TDS) and nitrate (NO3), while rising in dissolved oxygen (DO) content compared to untreated wastewater. Overall, this study suggests that phage-based treatment could be an effective and cheap alternative approach for the reduction of water pollutants in drainage drainage and waste water and thus eventually enhance the physicochemical quality of water.

Partial substitution of fish oil for linseed oil enhances beneficial fatty acids from rumen biohydrogenation but reduces ruminal fermentation and digestibility in growing goats.

This study was performed to investigate effects of partial replacement of fish oil (FO) for linseed oil (LO) on digestibility, ruminal fermentation and biohydrogenation in growing goats. Experiment 1 was carried out in four growing male goats aged 6 months in a 4 × 4 Latin square design. Goats were fed a basal diet supplemented with 25 g/kg dry matter either LO alone or in combination with tuna FO. Treatments were developed by replacing FO for LO at ratios of 0, 5, 10 and 15 g/kg DM corresponding to FO-0, FO-5, FO-10 and FO-15, respectively. Experiment 2 was carried out in an in vitro incubation system including 12 fermenters with the same four treatments. Each fermenter consisted of 40 mL goat ruminal fluid, 160 mL warm buffer, 2 g mixed substrates, and 50 mg FO-0, FO-5, FO-10 or FO-15. Fish oil inclusion reduced (P < 0.05) digestibility and nitrogen retention in Experiment 1. Increasing doses of FO in the diet induced a strong drop (P < 0.001) in ruminal total volatile fatty acid (VFA) concentration and protozoa population at 3 h post incubation, but did not affect individual VFA proportions. Substitution of FO for LO decreased mean concentrations of C18:0 (P = 0.057), c-9,c-12 C18:2 and C18:3n-3 (P < 0.001), but increased (P < 0.001) C20:5n-3 and C22:6n-3. Feeding FO-10 enhanced formation of ruminal c-9,t-11 conjugated linoleic acid (CLA) concentration compared with FO-0. Overall, combined data suggest that to improve ruminal concentrations of C20:5n-3, C22:6n-3, and c-9,t-11 CLA for deposition in tissues or milk with minimal risk of affecting digestibility and ruminal fermentation, a dietary supplementation of 15 g/kg LO and 10 g/kg FO would be suitable.