Master of Science

thesisGenetically encoded calcium indicators (GECIs) are Ca2+ sensitive fluorescent proteins that have expanded the usefulness of optical calcium imaging to longitudinal in vivo studies due to their advantage of direct expression in the tissue being imaged. Several generations of GECIs have been developed using green fluorescent protein (GFP) or one of its variants with each generation improving upon Ca2+-binding affinities and optical properties. However, the tissue penetration of excitation or emission light through tissue is small due to high absorption of available GECI wavelengths, which are shorter than the infrared range. The field still lacks a GECI with excitation or emission wavelengths in the infrared range, which has significantly less attenuation in biological tissue. Here we propose the development of an infrared GECI by insertion of the Ca2+-binding domain calmodulin (CaM) into regions surrounding the biliverdin chromophore binding pocket of infrared fluorescent protein (iRFP). We proposed seven DNA constructs of iRFP with different CaM insertion sites. Six of the seven DNA constructs were successfully produced with protein expressed from one of these constructs exhibiting similar optical properties to iRFP, showing successful receptor insertion into iRFP. Though our initial Ca2+ sensitivity test to monitor change in fluorescence due to Ca2+ binding is not conclusive, we open the field of GECI engineering to exciting new possibilities for noninvasive deep tissue calcium imaging

Draft genome sequence of the naphthalene degrader Herbaspirillum sp. strain RV1423

Herbaspirillum sp. strain RV1423 was isolated from a site contaminated with alkanes and aromatic compounds and harbors the complete pathway for naphthalene degradation. The new features found in RV1423 increase considerably the versatility and the catabolic potential of a genus of bacteria previously considered mainly to be diazotrophic endophytes to plants

The Impact of Dietary Zinc Oxide on the Bacterial Diversity of the Small Intestinal Microbiota of Weaned Piglets

Dietary zinc oxide is often used in pharmacological concentrations to promote health as well as performance of weaned piglets due to its bacteriostatic effects. This study was conducted to provide an in depth analysis of the bacterial composition in weaned piglets fed different amounts of dietary zinc oxide. Piglets were fed diets containing 57 (low), 164 (medium) or 2425 (high) mg/kg dietary zinc. Zinc above the basal dietary level was supplied from analytical grade zinc oxide (ZnO). DNA was extracted from stomach and ileum digesta samples of 32 and 53d old animals (n=4 per group) and used to generate bar-coded 16S ribosomal DNA amplicons for deep sequencing analysis. A total of 9 phyla, 40 orders, 75 families and 328 genera were detected in 8.76 x 105 sequencing reads. Firmicutes, Bacteroidetes and Proteobacteria were the dominant phyla, but no significant differences between treatment groups were observed. Lactobacillales (16.3-59.9%), Bacteroidales (2.2-59.1%), Clostridiales (0.05-70.2%) and Selenomonadales (2.6-17.5%) were found as the dominating order. Noteworthy changes on the order level were found for numerically or significantly increased ratios of Clostridiales, but significantly decreased Lactobacillales in the high dietary zinc group. The bacterial diversity for the high dietary zinc diet was significantly higher for the total microbiota than the medium or low zinc diet. However, Lactobacillales diversity decreased, while Clostridiales and Enterobacteriales diversity increased significantly. Principal component analysis confirmed changes in the microbiota, most notably for the high dietary zinc treatment. This study has shown that pharmacological doses of high dietary zinc can drastically alter the bacterial composition and development of the microbiota in weaned piglets. The quantitative shift of bacterial groups due to high dietary zinc was most pronounced one week after weaning, while the more developed microbiota in older animals seemed to be able to adapt to high concentrations of dietary zinc

Dietary bioactive lipid compounds rich in menthol alter Interactions among members of ruminal microbiota in sheep

This study aimed to investigate the effects of two practically relevant doses of menthol-rich plant bioactive lipid compounds (PBLC) on fermentation, microbial community composition, and their interactions in sheep rumen. Twenty-four growing Suffolk sheep were divided into three treatments and were fed hay ad libitum plus 600 g/d of concentrate containing no PBLC (Control) or PBLC at low dose (80 mg/d; PBLC-L) or high dose (160 mg/d; PBLC-H). After 4 weeks on the diets, samples of ruminal digesta were collected and analyzed for short-chain fatty acid (SCFA), ammonia, and microbiota; microbiota being analyzed in the solid and the liquid digesta fractions separately. Ruminal SCFA and ammonia concentrations were not affected by the PBLC treatments. The microbiota in the solid fraction was more diverse than that in the liquid fraction, and the relative abundance of most taxa differed between these two fractions. In the solid fraction, phylogenetic diversity increased linearly with increased PBLC doses, whereas evenness (lowest in PBLC-L) and Simpson diversity index (greatest in PBLC-H) changed quadratically. In the liquid fraction, however, the PBLC supplementation did not affect any of the microbial diversity measurements. Among phyla, Chloroflexi (highest in PBLC-L) and unclassified_bacteria (lowest in PBLC-L) were altered quadratically by PBLC. Lachnospiraceae, Bacteroidaceae (increased linearly), BS11 (increased in PBLC-L), Christensenellaceae (decreased in PBLC treatments), and Porphyromonadaceae (increased in PBLC treatments) were affected at the family level. Among genera, Butyrivibrio increased linearly in the solid fraction, YRC22 increased linearly in the liquid fraction, whereas Paludibacter increased and BF311 increased linearly with increasing doses of PBLC in both fractions. The PBLC treatments also lowered methanogens within the classes Thermoplasmata and Euryarchaeota. Correlation network analysis revealed positive and negative correlations among many microbial taxa. Differential network analysis showed that PBLC supplementation changed the correlation between some microbial taxa and SCFA. The majority of the predicted functional features were different between the solid and the liquid digesta fractions, whereas the PBLC treatments altered few of the predicted functional gene categories. Overall, dietary PBLC treatments had little influence on the ruminal fermentation and microbiota but affected the associations among some microbial taxa and SCFA

Porcine Colostrum Protects the IPEC-J2 Cells and Piglet Colon Epithelium against Clostridioides (syn. Clostridium) difficile Toxin-Induced Effects

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet’s intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring

Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells

Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features

Local chiral potentials and the structure of light nuclei

We present fully local versions of the minimally non-local nucleon-nucleon potentials constructed in a previous paper [M.\ Piarulli {\it et al.}, Phys.\ Rev.\ C {\bf 91}, 024003 (2015)], and use them in hypersperical-harmonics and quantum Monte Carlo calculations of ground and excited states of $^3$H, $^3$He, $^4$He, $^6$He, and $^6$Li nuclei. The long-range part of these local potentials includes one- and two-pion exchange contributions without and with $\Delta$-isobars in the intermediate states up to order $Q^3$ ($Q$ denotes generically the low momentum scale) in the chiral expansion, while the short-range part consists of contact interactions up to order $Q^4$. The low-energy constants multiplying these contact interactions are fitted to the 2013 Granada database in two different ranges of laboratory energies, either 0--125 MeV or 0--200 MeV, and to the deuteron binding energy and $nn$ singlet scattering length. Fits to these data are performed for three models characterized by long- and short-range cutoffs, $R_{\rm L}$ and $R_{\rm S}$ respectively, ranging from $(R_{\rm L},R_{\rm S})=(1.2,0.8)$ fm down to $(0.8,0.6)$ fm. The long-range (short-range) cutoff regularizes the one- and two-pion exchange (contact) part of the potential.Comment: 29 pages, 3 figure

Performance considerations for the application of the lossless browse and residual model

A hybrid lossless compression model employing both the (lossy) JPEG DCT algorithm and one of a selection of lossless image compression methods has been tested. The hybrid model decomposes the original image into a low-loss quick-look browse and a residual image. The lossless compression methods tested in the model are Huffman, arithmetic, LZW, lossless JPEG, and diagonal coding. For both the direct and the hybrid application of these lossless methods, the compression ratios (CR's) are calculated and compared on three test images. For each lossless method tested, the hybrid model had no more than a nominal loss in compression efficiency relative to the direct approach. In many cases, the hybrid model provided a significant compression gain. When used in the hybrid model, lossless JPEG outperformed the other lossless methods over a broad range of browse image qualities.Approved for public release; distribution is unlimited