Analysis of the cells isolated from epithelial cell rests of Malassez through single-cell limiting dilution

The epithelial cell rests of Malassez (ERM) are essential in preventing ankylosis between the alveolar bone and the tooth (dentoalveolar ankylosis). Despite extensive research, the mechanism by which ERM cells suppress ankylosis remains uncertain; perhaps its varied population is to reason. Therefore, in this study, eighteen unique clones of ERM (CRUDE) were isolated using the single-cell limiting dilution and designated as ERM 1–18. qRT-PCR, ELISA, and western blot analyses revealed that ERM-2 and -3 had the highest and lowest amelogenin expression, respectively. Mineralization of human periodontal ligament fibroblasts (HPDLF) was reduced in vitro co-culture with CRUDE ERM, ERM-2, and -3 cells, but recovered when an anti-amelogenin antibody was introduced. Transplanted rat molars grown in ERM-2 cell supernatants produced substantially less bone than those cultured in other cell supernatants; inhibition was rescued when an anti-amelogenin antibody was added to the supernatants. Anti-Osterix antibody staining was used to confirm the development of new bones. In addition, next-generation sequencing (NGS) data were analysed to discover genes related to the distinct roles of CRUDE ERM, ERM-2, and ERM-3. According to this study, amelogenin produced by ERM cells helps to prevent dentoalveolar ankylosis and maintain periodontal ligament (PDL) space, depending on their clonal diversity.

Development of a Serial Dilution Technique for Obtaining Monoclonal Cell Populations

Single cell-based techniques have drawn the attention of researchers, because they provide invaluable information of various domains ranging from genomics to epigenetics, transcriptomics, and proteomics. Single cell-derived clones provide a reliable and sustainable source of genetic information due to the homogeneity of the cell population. Aiming to obtain single-cell clones, several approaches were engineered, among which, the Limiting dilution approach stands out as a cost-effective and unsophisticated, and easy-to-apply method. Here, we demonstrate how to acquire single cell-derived clones through a simple 1:10 diluting from genetically modified heterogeneous cell populations.

An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection

Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.

Improved Plasmodium falciparum dilution cloning through efficient quantification of parasite numbers and c-SNARF detection

Background Molecular and genetic studies of blood-stage Plasmodium falciparum parasites require limiting dilution cloning and prolonged cultivation in microplates. The entire process is laborious and subject to errors due to inaccurate dilutions at the onset and failed detection of parasite growth in individual microplate wells. Methods To precisely control the number of parasites dispensed into each microplate well, parasitaemia and total cell counts were determined by flow cytometry using parasite cultures stained with ethidium bromide or SYBR Green I. Microplates were seeded with 0.2 or 0.3 infected cells/well and cultivated with fresh erythrocytes. The c-SNARF fluorescent pH indicator was then used to reliably detect parasite growth. Results Flow cytometry required less time than the traditional approach of estimating parasitaemia and cell numbers by microscopic examination. The resulting dilutions matched predictions from Poisson distribution calculations and yielded clonal lines. Addition of c-SNARF to media permitted rapid detection of parasite growth in microplate wells with high confidence. Conclusion The combined use of flow cytometry for precise dilution and the c-SNARF method for detection of growth improves limiting dilution cloning of P. falciparum. This simple approach saves time, is scalable, and maximizes identification of desired parasite clones. It will facilitate DNA transfection studies and isolation of parasite clones from ex vivo blood samples.

Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA

The presence of a stable HIV-1 reservoir persisting over time despite effective antiretroviral suppression therapy precludes a cure for HIV-1. Characterizing and quantifying this residual reservoir is considered an essential prerequisite to develop and validate curative strategies. However, a sensitive, reproducible, cost-effective, and easily executable test is still needed. The quantitative viral outgrowth assay is considered the gold standard approach to quantify the reservoir in HIV-1-infected patients on suppressive ART, but it has several limitations. An alternative method to quantify the viral reservoir following the reactivation of latent HIV-1 provirus detects multiply-spliced tat/rev RNA (msRNA) molecules by real-time PCR [tat/rev induced limiting dilution assay (TILDA)]. This article provides a perspective overview of the clinical relevance, various applications, recent advancements of TILDA, and how the assay has contributed to our understanding of the HIV-1 reservoir.

Analysis of The Cells Isolated From Epithelial Cell Rests of Malassez Through Single-cell Limiting Dilution

The epithelial cell rests of Malassez (ERM) play a pivotal role in preventing ankylosis between the alveolar bone and the tooth. Although several functions of ERM has been reported, the mechanism behind preventing dentoalveolar ankylosis remains unclear. In this study, 18 clones were isolated from ERM (CRUDE) using the single-cell limiting dilution method. Among them, ERM-2 and -3, which exhibited the highest and lowest proliferation rates, respectively, were selected. ERM-2, ERM-3, and CRUDE ERM were stained with epithelial markers, including cytokeratin-wide and cytokeratin-19, via immunofluorescence. The qRT-PCR analysis revealed increased expression levels of p75 (ameloblast marker), amelogenin, and sfrp5 (inner enamel epithelial cell marker) in the ERM-2 cells. Alternatively, ameloblastin and ck-14 (outer enamel epithelial cell marker) were highly expressed in ERM-3 cells. The mineralization of human periodontal ligament fibroblast (HPDLF) was inhibited when co-cultured with ERM-2, ERM-3, and CRUDE ERM cells. The addition of an anti-amelogenin antibody restored the mineralization of HPDLF cells. Transplanted rat molar cultured in ERM-2 (high amelogenin secretive clone) cell-derived supernatant resulted in significantly smaller bone formation than those cultured in the CRUDE ERM and ERM-3 cell-derived supernatants. These findings indicate that amelogenin produced by ERM cells might be involved in preventing dentoalveolar ankylosis.

#89: Lytic Bacteriophage Against an Emergent Biofilm-Forming Enteroinvasive Escherichia coli

Background Bacteriophage are viruses that infect and parasitize bacteria. In the pre-antibiotic era, phage therapy was one of the few tools available against bacterial infection. After penicillin was discovered, phage therapy fell into disfavor in Western medicine, and its use was mostly limited to medical centers in the Soviet bloc. With current issues on multi-drug resistance, especially of Gram-negative bacteria, there is interest in revisiting phage therapy for use in medical and veterinary practice. This study focuses on the biofilm-forming enteroinvasive Escherichia coli (BF-EIEC) serotype O96:H19. Strain BF-EIEC 52.1 was isolated in a pediatric case–control diarrheal study in Bucaramanga, Colombia, in 2013–2014. This strain is genetically similar to a foodborne outbreak strain isolated in Italy in 2012, and in sporadic outbreaks in the UK, Spain, and Uruguay. BF-EIEC 52.1 produces biofilms in vitro, similarly to enteroaggregative E. coli, and yet invades host cells, similar to enteroinvasive E. coli and Salmonella enterica (J. Iqbal, O. Gómez-Duarte et al., manuscript in preparation), making this a potential emergent pathogen of concern. We hypothesized that lytic bacteriophage derived from wastewater sources are active against BF-EIEC 52.1. Methods Environmental wastewater samples were retrieved from the Amherst, New York, wastewater treatment facility. These were filtered against a 0.2 μm membrane and stored at 4°C. An attempt to isolate phage was done by direct plating over E. coli BF-EIEC 52.1 on solid Luria–Bertani (LB) agar. A second strategy involved enrichment for lytic phage by incubating sample water with BF-EIEC 52.1 in LB broth. Cloning was accomplished by re-filtering the preparation (to remove bacteria), then followed by serial limiting dilution of cell-free filtrate in liquid bacterial culture. Characterization of phage preparations included a demonstration of differential lytic activity against BF-EIEC 52.1 and two unrelated laboratory E. coli strains, DH5α and HB101, in both solid agar and liquid medium. Results Isolation of phage by direct plating was possible, but cloning phages proved difficult. With the alternative strategy, four of six wastewater specimens incubated with BF-EIEC 52.1 in liquid LB showed decreased turbidity compared with sterile water control. All positive specimens proved to have demonstrable lytic activity against host E. coli by plaque formation in solid medium, and in limiting dilution in liquid medium. Thirty-two putative phage preparations were cloned by repeated limiting dilution from an initial 16 independent wells. Of these, 27 preparations showed visible plaques against BF-EIEC 52.1 in solid agar, while 29 and 23 preparations showed plaques against HB101 and DH5α, respectively. All phage preparations inhibited the growth of low-inoculum BF-EIEC 52.1 in liquid medium for >6 hours. Six virus preparations inhibited growth for >72 hours. Conclusions Bacteriophage active against strain BF-EIEC 52.1 were isolated from urban wastewater samples, showing a range of activity against the parental host, as well as unrelated E. coli strains. Further study is needed to demonstrate activity in alternative clinical settings, such as in biofilm-associated infections or in animal models.

A Modified Limiting Dilution Method for Monoclonal Stable Cell Line Selection Using a Real-Time Fluorescence Imaging System: A Practical Workflow and Advanced Applications

Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte®S3.