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Since the onset of molecular biology, the ciliate Tetrahymena thermophila has been one of the most convenient single-celled model eukaryotes for genetics, biochemistry, and cell biology. Particularly, thanks to the availability of several different selection markers, it is possible to knock out or knock in genes at multiple genetic loci simultaneously in Tetrahymena, which makes it an excellent model ciliate for tackling complex regulatory mechanisms. Despite these selection markers are efficient for genetic manipulation, the costly drugs used for selection have highlighted the urgent demand for an additional cost-efficient and effective selection marker. Here, we found that a mutated 60S ribosomal subunit component, RPL36A, confers Tetrahymena with cycloheximide resistance. On top of that, we developed a cycloheximide cassette and explored suitable transformation and selection conditions. Using the new cassette, we obtained both knock-out and knock-in strains successfully at a relatively low cost. This study also provided the first evidence that a cycloheximide resistance gene can be engineered as a selection marker to completely delete a gene from the highly-polyploid somatic nucleus in Tetrahymena.

In this study, a co-culturing Enterobacter sp. and Lactococcus lactis strategy was developed to alter bacterial cellulose (BC) properties and increase nisin yields. We generated high nisin yields (6260 IU/mL) by altering inoculum ratios and inoculation times in a novel co-culture system. Critically, these were 85% higher than L. lactis monocultures. By monitoring fermentation broth pH and lactic acid yields, the pH was higher and lactic acid yields lower during co-culture conditions when compared with L. lactis monocultures, suggesting that co-culturing was more suitable for L. lactis nisin production. We also determined BC film yields and properties (BC, BC-N, and BC-N after nisin release). BC yields produced by co-culturing were not very different from Enterobacter sp. monocultures, but crystallinity was significantly altered. Collectively, our co-culture system adequately and economically modified BC fibers by interfering with self-assembly and crystallization processes during BC synthesis, with significantly improved nisin yields.

Gel properties of hydrogel-forming by Ala-Lys dipeptide (AK) and iota-carrageenan (ι-C) were investigated by rheological behavior, fourier transform infrared analysis, cryo-scanning electron microscopy, low field-NMR relaxometry and magnetic resonance imaging. Iota-carrageenan was changed from a liquid to a gel with the addition of AK, and the existence of AK significantly increased the storage modulus (G') of ι-C from 590.4 to 1077.8 Pa. In the ι-C/AK gel, the blue-shift of OH stretching and water deformation were observed, meanwhile, the presence of amide I band at 1682 cm-1 was observed. The network of ι-C/AK gel showed a dense honeycomb structure with flocculating continuous phase and rough entanglement morphology. After adding AK, the water free in the pores of ι-C entered the ι-C/AK gel matrix, and the binding capacity of bound water was enhanced. These scenarios proved that the AK as the cationic dipeptide could control the conversion of negatively charged ι-C from an original random structure to a helical structure due to electrostatic interactions and hydrogen bonds. This study provides a new opportunity for the peptides into carbohydrate-based gel matrices, which could provide insights for the further application of ι-C/AK gels in the fields of food industry, tissue engineering and drug delivery.

MicroRNAs (miRNAs) have been shown to be important regulators in the pathogenesis of osteoarthritis (OA). The objective of this study was to determine the expression levels of miR-483, miR-483*, their host gene (Igf2) and other cytokines in a murine model of OA. The expression of miR-483 was significantly up-regulated in old mouse and in all of the operation groups, particularly the group assessed 1 week after surgery. The expression of miR-483* was significantly increased in the old mouse group and the group assessed 5 weeks after surgery. The expression of miR-483 was negatively correlated with the expression of (mRNA) Bmp7 and TgfB and positively correlated with Mmp13 by Pearson correlation analysis, while miR-483* was positively correlated with Il1B. Surprisingly, there was no correlation between the expression of either miR-483 or miR-483* and Igf2. This study shows that the expression of miR-483 and miR-483* is up-regulated in murine OA. These data suggest that miR-483 and miR-483* may play critical roles in early and later pathogenesis of OA, respectively, without the involvement of their host gene Igf2.