Mazumdar, M. | bioRxiv Science

bioRxiv2022

Use of Solentim verified in-situ plate seeding (VIPSTM) enhances single-cell cloning efficiency

The primary goal in cell line development is to establish high-producer recombinant cell line(s) of single-cell origin. Traditionally, these cell lines are developed using limiting dilution cloning (LDC) method of single cell isolation, a rate-limiting, lengthy and labor-intensive process. The Verified-In-Situ-Plate-Seeding (VIPS) is an automated single-cell seeding and imaging equipment designed to accelerate cell line development workflow. In this study, VIPS was tested for efficiency and accuracy of single cell seeding in parallel with limiting dilution cloning (LDC). Three Chinese hamster ovary (CHO) derived cell lines with known clonal properties were tested under six different growth conditions (three growth media and two different kinds of microplates). Data showed VIPS and limiting dilution (LDC) have comparable cloning efficiency when CHO-M cells were tested. By contrast, the Verified In-Situ Plate Seeding (VIPS) produced 6-8-fold more clones of single-cell origin than LDC when CHO-K1 or CHO-S cells were tested. Moreover, the verified In-Situ Plate Seeding (VIPS) correctly identified single-cell and multiple-cells seeded wells with 65-72% and 52-81% accuracy, respectively. Taken together, the high throughput imaging and single-cell seeding capabilities of VIPS outperformed the rate-limiting LDC method and, therefore, has the potential to accelerate cell line development workflow.

cell biology↗

bioRxiv2020

Queuine is a nutritional regulator of Entamoeba histolytica response to oxidative stress and a virulence attenuator

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here we describe the effects of queuine on the physiology of the eukaryotic parasite, Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in tRNAAspGUC. Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in OS response, such as heat shock protein 70 (Hsp 70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in tRNAAspGUC, parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence. ImportanceEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of the bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces a mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

microbiology↗