In Vivo Non-Ribosomal Protein Synthesis in Mammalian Cells

The mechanism of Sortase A substrate specificity has been widely studied and applied to many approaches to bioconjugation. Current research includes transpeptidation between peptide nucleotide acids, polypeptides, viruses, or antibodies. These applications help improve drug targeting and delivery. Scientists have performed Sortase A-mediated protein ligation in vitro. This project proposes an in vivo protein ligation method with Sortase A. In this design, Sortase A acts as a catalyst to initiate bioconjugation between the LPETG motif and pentaglycine (Gly5) chain to express GFP. This technique bypasses the ribosome and offers an alternative way to synthesize protein in mammalian cells. Three recombinant plasmids were created with the GFP gene spliced into two segments. The LPETG motif is inserted to the N-terminus of GFP and the Gly5 chain is attached to the C-terminus of GFP. The recombinant plasmids are co-transfected into HeLa cells with another recombinant plasmids that expresses only for Sortase A. The expression of GFP is observed under a fluorescence microscope every 12 hours. Detection of GFP by fluorescence indicates the reconstruction of whole GFP via in vivo protein ligation is successful. Overall, this design pioneers synthetic biology by reengineering cellular pathways in a mammalian cell. There is potential to create synthetic cells and apply Sortase A to trigger in vivo ligation between a protein drug and a drug target for precision medicine

Performance of torus-type brushless DC motor with winding connected in two and three-phase system

A torus-type permanent magnet brushless DC motor is the object of this thesis. The motor can operate with the winding connected either in two - phase or three - phase system. A comparative study of performance of the motor operating in these two systems is the objective of the thesis. To analyze the motor performance, the dynamic models of the motors operating with two - phase and three - phase motors have been proposed. The simulation of motor operation was carried out using the MATLAB/SIMULINK software package. A study of motors operation in steady state conditions has been done by applying simpler models which were based on brush DC motor equivalent circuit. It is observed that the electromechanical characteristics of these two motors are similar and the motor with three - phase winding has lesser quantity of torque ripples. Since the motors operating in the two types winding connection systems are supplied from voltage - type inverters, switching conditions have a significant influence on motor performance. This was also studied in the thesis project using the dynamic motor model. The results of this study show that by advancing the commutation angle, the efficiencies could be improved greatly

Spatial Sensor Network Based Target Tracking By Classification

The wide use of sensor networks in the day to day communication in recent trends made tracking a significant feature in monitoring systems. The automated systems capable of detection and tracking of targets is a desirable application in many fields. Firstly, deploy a sensor network with appropriate space between sensors and then introduce targets into the network. As the sensors detect the targets, each sensor communicates with neighborhood sensor nodes and one of those sensors are elected as cell-head which will calculate the position of target from the data and transmit that to sink. This process is repeated iteratively to track the moving target. Feature extraction methods and classification techniques have been studied to classify targets by their type. For the challenging task of Multi-target tracking, the methods of sequential Bayesian filtering and Sequential Monte Carlo-Particle Hypothesis Density filters are sought. Accurate algorithms have been simulated for Localization and tracking of target using the data of sensor strengths which are collaboratively communicated among the sensors. Direction of moving target inside a cell was estimated. Algorithm for Hierarchical multi-hop communication model was established

Effects of Eimeria maxima infection doses on growth performance and gut health in dual-infection model of necrotic enteritis in broiler chickens

The objective of this study was to investigate the effects of the different doses of Eimeria maxima (EM) oocysts on growth performance and intestinal health in broiler chickens challenged with a dual infection model of necrotic enteritis (NE) using EM and NetB+Clostridium perfringens (CP). A total of 432 fourteen-d-old male Cobb 500 broiler chickens were divided into 6 groups with 6 replicates each. The six different groups were as follows: Control, non-challenged; T0+, challenged with CP at 1 × 109 colony forming unit; T5K+, T0+ + 5,000 EM oocysts; T10K+, T0+ + 10,000 EM oocysts; T20K+; T0+ + 20,000 EM oocysts; and T40K+; T0+ + 40,000 EM oocysts. The challenge groups were orally inoculated with EM strain 41A on d 14, followed by NetB+CP strain Del-1 on 4 days post inoculation (dpi). Increasing EM oocysts decreased d 21 body weight, body weight gain, feed intake (linear and quadratic, p < 0.001), and feed efficiency (linear, p < 0.001) from 0 to 7 dpi. Increasing EM oocysts increased jejunal NE lesion score and intestinal permeability on 5, 6, and 7 dpi (linear, p < 0.05). On 7 dpi, increasing the infection doses of EM oocysts increased jejunal CP colony counts (linear, p < 0.05) and increased fecal EM oocyst output (linear and quadratic, p < 0.001). Furthermore, increasing the infection doses of EM oocysts decreased the villus height to crypt depth ratios and the goblet cell counts (linear, p < 0.05) on 6 dpi. Increasing EM oocysts downregulated the expression of MUC2, B0AT, B0,+AT, PepT1, GLUT2, AvBD3 and 9, LEAP2, and TLR4, while upregulating CLDN1, CATHL3, IL-1β, IFN-γ, TNFSF15, TNF-α, IL-10, and Gam56 and 82 on 6 dpi (linear, p < 0.05). Additionally, increasing EM oocysts decreased Pielou’s evenness and Shannon’s entropy (linear, p < 0.01). In conclusion, increasing the infection doses of EM significantly aggravated the severity of NE and exerted negative impact on intestinal health from 5 to 7 dpi

Surface Enhanced Raman Scattering and Gated Materials for Sensing Applications: The Ultrasensitive Detection of Mycoplasma and Cocaine

[EN] We present herein a novel combination of gated mesoporous silica nanoparticles (MSNs) and surface-enhanced Raman scattering (SERS) for sensing applications. As a proof-of-concept, we show the design of a system comprising MSNs loaded with crystal violet (CV), a molecule with high Raman cross section acting as SERS reporter, and capped with either a suitable DNA sequence for the detection of Mycoplasma genomic DNA or with an aptamer that selectively coordinates cocaine. In both cases the presence of the corresponding target analyte in solution (i.e., genomic DNA or cocaine) resulted in the release of CV. CV delivery was detected by SERS upon adsorption on gold nanotriangles (AuNTs), which display an efficient electromagnetic field enhancement and a high colloidal stability. By using this novel procedure a limit of detection of at least 30 copies DNA per mL was determined for the detection of Mycoplasma genomic DNA, whereas cocaine was detected at concentrations as low as 10 nm.M.C.-P. acknowledges an FPU Scholarship from the Spanish Ministry of Education, Culture and Sports. L.M.L.- M. acknowledges financial support from the European Research Council (ERC Advanced Grant #267867 Plasmaquo) and the European Union's Seventh Framework Programme (FP7/2007-2013 under Grant Agreement No 312184, SACS). M.O. is grateful to the Universitat Politecnica de Valencia for a FPI-UPV grant. Financial support from the Spanish Government (Project MAT2015-64139-C4-1-R MINECO/FEDER) and the Generalitat Valenciana (Project PROMETEOII/2014/047) is gratefully acknowledged.Oroval, M.; Coronado Puchau, M.; Langer, J.; Sanz-Ortiz, MN.; Ribes, À.; Aznar, E.; Coll Merino, MC.... (2016). Surface Enhanced Raman Scattering and Gated Materials for Sensing Applications: The Ultrasensitive Detection of Mycoplasma and Cocaine. Chemistry - A European Journal. 22(38):13488-13495. https://doi.org/10.1002/chem.201602457S1348813495223

Complexity of the Mycoplasma fermentans M64 Genome and Metabolic Essentiality and Diversity among Mycoplasmas

Recently, the genomes of two Mycoplasma fermentans strains, namely M64 and JER, have been completely sequenced. Gross comparison indicated that the genome of M64 is significantly bigger than the other strain and the difference is mainly contributed by the repetitive sequences including seven families of simple and complex transposable elements ranging from 973 to 23,778 bps. Analysis of these repeats resulted in the identification of a new distinct family of Integrative Conjugal Elements of M. fermentans, designated as ICEF-III. Using the concept of “reaction connectivity”, the metabolic capabilities in M. fermentans manifested by the complete and partial connected biomodules were revealed. A comparison of the reported M. pulmonis, M. arthritidis, M. genitalium, B. subtilis, and E. coli essential genes and the genes predicted from the M64 genome indicated that more than 73% of the Mycoplasmas essential genes are preserved in M. fermentans. Further examination of the highly and partly connected reactions by a novel combinatorial phylogenetic tree, metabolic network, and essential gene analysis indicated that some of the pathways (e.g. purine and pyrimidine metabolisms) with partial connected reactions may be important for the conversions of intermediate metabolites. Taken together, in light of systems and network analyses, the diversity among the Mycoplasma species was manifested on the variations of their limited metabolic abilities during evolution

n-3 enriched Fish oil diet enhanced intestinal barrier integrity in broilers after Eimeria infection

ABSTRACT: Coccidiosis caused by Eimeria spp. results in substantial economic losses in the poultry industry. The objective of this study was to investigate the effects of dietary supplementation with n-3 polyunsaturated fatty acids-enriched fish oil on growth performance, intestinal barrier integrity, and intestinal immune response of broilers challenged with Eimeria spp. A total of 576 fourteen-day-old broilers were randomly assigned in a completely randomized design with a 3 × 2 factorial arrangement, comprising 2 diets supplemented with either 5% fish oil or 5% soybean oil, and 3 Eimeria spp. infection levels: a nonchallenge control, a low dose of Eimeria challenge, and a high challenge dose. The results of the study revealed significant interactions between diet and Eimeria challenge to parameters of gut barrier integrity and feed intake. A significant interaction was observed in feed intake between 5 and 8 d postinfection (DPI), where the fish oil groups exhibited a higher amount of feed intake compared to the soybean oil diet groups after coccidiosis infection. The effects of the fish oil diet resulted in enhanced gut barrier integrity, as evidenced by a trend of decreased gastrointestinal leakage and a lower mean of small intestine lesion scores after Eimeria challenge. Additionally, significant interactions were noted between Eimeria spp. challenge and diet regarding jejunal crypt depth. The positive impact of the fish oil diet was particularly noticeable with the high Eimeria challenge dose. Overall, these findings underscore the relationship between the fish oil diet and Eimeria challenge on broiler chicken intestinal health. Dietary supplementation of fish oil has the potential to maintain small intestine barrier integrity with severe Eimeria infection conditions

Altered Osteogenic Differentiation in Mesenchymal Stem Cells Isolated from Compact Bone of Chicken Treated with Varying Doses of Lipopolysaccharides

Persistent inflammation biologically alters signaling molecules and ultimately affects osteogenic differentiation, including in modern-day broilers with unique physiology. Lipopolysaccharides (LPS) are Gram-negative bacterial components that activate cells via transmembrane receptor activation and other molecules. Previous studies have shown several pathways associated with osteogenic inductive ability, but the pathway has yet to be deciphered, and data related to its dose-dependent effect are limited. Primary mesenchymal stem cells (MSCs) were isolated from the bones of day-old broiler chickens, and the current study focused on the dose-dependent variation (3.125 micrograms/mL to 50 micrograms/mL) in osteogenic differentiation and the associated biomarkers in primary MSCs. The doses in this study were determined using a cell viability (MTT) assay. The study revealed that osteogenic differentiation varied with dose, and the cells exposed to higher doses of LPS were viable but lacked differentiating ability. However, this effect became transient with lower doses, and this phenotypic character was observed with differential staining methods like Alizarin Red, Von Kossa, and alkaline phosphatase. The data from this study revealed that LPS at varying doses had a varying effect on osteogenic differentiation via several pathways acting simultaneously during bone development