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Abstract The role of the mechanical environment in defining tissue function, development and growth has been shown to be fundamental. Assessment of the changes in stiffness of tissue matrices at multiple scales has relied mostly on invasive and often specialist equipment such as AFM or mechanical testing devices poorly suited to the cell culture workflow.In this paper, we have developed a unbiased passive optical coherence elastography method, exploiting ambient vibrations in the sample that enables real-time noninvasive quantitative profiling of cells and tissues. We demonstrate a robust method that decouples optical scattering and mechanical properties by actively compensating for scattering associated noise bias and reducing variance. The efficiency for the method to retrieve ground truth is validated in silico and in vitro, and exemplified for key applications such as time course mechanical profiling of bone and cartilage spheroids, tissue engineering cancer models, tissue repair models and single cell. Our method is readily implementable with any commercial optical coherence tomography system without any hardware modifications, and thus offers a breakthrough in on-line tissue mechanical assessment of spatial mechanical properties for organoids, soft tissues and tissue engineering.

Summary: LINE1 is the most active and abundant family of retrotransposons; it is implicated in a number of pathologies, as well as in early embryo development. We present a protocol to specifically knockdown LINE1 in mouse embryonic stem cells and embryos, including details for the nucleofection and zygote microinjection of LINE antisense oligos, followed by RNA FISH validation. This protocol can be used in development, as well as other cell types where LINE1 is believed to be expressed.For complete information on the use and execution of this protocol, please refer to Percharde et al. (2018).

Although laser irradiation and implantoplasty (IP) are both treatment options for peri-implantitis, no studies have yet combined these two treatment solutions. The aim of this study was to identify the effect of an Er, Cr: YSGG laser on the IP surface. In experiment 1, TiUnite anodized surface implants were treated with an Er, Cr: YSGG laser at 0.5 to 2 W on the panel energy setting and 20 Hz under water irrigation. In experiment 2, all implant surfaces were treated with the IP procedure first, then irradiated with the Er, Cr: YSGG laser. All samples were analyzed by stereomicroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and surface topography. Stereomicroscopy and SEM revealed no obvious surface change at any energy setting once the surface was polished with the IP procedure, whereas damage was caused to the TiUnite original implant surface when the Er, Cr: YSGG laser panel energy was set at 1 W or higher. EDS showed no significant difference in element composition once the surface was polished with the IP procedure, while a compositional change was detected when the Er, Cr: YSGG laser panel energy was set to 0.5 W or higher to irradiate the original TiUnite surface. Surface roughness may be related to laser irradiation energy, but no significant changes occurred following IP. These results indicated that the Er, Cr: YSGG laser may have little effect on the post-IP surface compared with the virgin TiUnite surface.

Background/Purpose: Physicians' and dentists' knowledge of levamisole-induced agranulocytosis or pancytopenia remains incomplete. This study aimed to evaluate the treatment effectiveness of levamisole plus prednisolone on oral lichen planus (OLP) patients with emphasis on levamisole-induced hematological changes. Methods: Ninety patients with erosive OLP were given 120 mg/day new levamisole (Levazol) and 15 mg/day prednisolone for three consecutive days each week. Three cases with levamisole-induced blood-cytopenias were assessed and treated within one year. Results: Most patients reported significant pain relief and showed no evidence of erosive OLP after 4–8 weeks of treatment with few side effects; nevertheless, three female patients developed agranulocytosis or granulocytopenia with concomitant thrombocytopenia or pancytopenia within 2–6 weeks after levamisole (Levazol) treatment. One case with previously unknown double episodes of agranulocytosis revealed her first episode following interruption of levamisole (Decaris) treatment for 4 months. High fever and sore throat were the most common symptoms, but two agranulocytosis cases remained asymptomatic one week before diagnosis, and were treated with levamisole withdrawal and empiric antimicrobial initiation as well as utilization of granulocyte colony-stimulating factors. Neutrophil recovery took about 1 week, but over 4 weeks in one of the cases (an elderly patient) with septic shock. Conclusion: Agranulocytosis or pancytopenia usually developed within 2 months after levamisole treatment, but it might be delayed. Agranulocytosis was more likely to occur in females and onset was acute. Levamisole is an effective immunomodulator for OLP patients; however, it should be used with caution and administered with regular blood monitoring. Keywords: Levamisole, Agranulocytosis, Pancytopenia, Oral lichen planus

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)—a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

The ability of small molecules to maintain self-renewal and to inhibit differentiation of pluripotent stem cells has been well-demonstrated. Two widely used molecules are PD 98059 (PD), an inhibitor of extracellular-signal-regulated kinase 1 (ERK), and SC1 (Pluripotin), which inhibits the RasGAP and ERK pathways. However, no studies have been conducted to compare their effects on the pluripotency and derivation of embryonic stem (ES) cells from inbred mice C57BL/6, an important mouse strain frequently used to model behavior, cognitive functions, immune system, and metabolic disorders in humans and also the first mouse strain chosen to be sequenced for its entire genome. We found significantly increased derivation efficiency of ES cells from in vivo fertilized embryos (fES) of C57BL/6 with the use of PD (71.4% over the control of 35.3%). Because fES and ES from cloned embryos (ntES) are not distinguishable in transcription or translation profiles, we used ntES cells to compare the effect of small molecules on their in vitro characteristics, in vitro differentiation ability, and the ability to generate full-term ntES-4N pups by tetraploid complementation. NtES cells exhibited typical ES characteristics and up-regulated Sox2 expression in media with either small-molecule. Higher rates of full term ntES-4N pup were generated by the supplementation of PD or SC1. We obtained the highest efficiency of ntES-4N pup generation ever reported from this strain by supplementing ES medium with SC1. Lastly, we compared the pluripotency of fES, ntES and induced pluripotent stem (iPS) cells of C57BL/6 background using the tetraploid complementation assay. A significant increase in implantation sites and the number of full-term pups were obtained when fES, ntES, and iPS cells were cultured with SC1 compared to the control ES medium. In conclusion, supplementing ES cell culture medium with PD and SC1 increases the derivation efficiency and pluripotency, respectively, of stem cells derived from the refractory inbred C57BL/6 strain.