Beyond FOXP3:a 20-year journey unravelling human regulatory T-cell heterogeneity

The initial idea of a distinct group of T-cells responsible for suppressing immune responses was first postulated half a century ago. However, it is only in the last three decades that we have identified what we now term regulatory T-cells (Tregs), and subsequently elucidated and crystallized our understanding of them. Human Tregs have emerged as essential to immune tolerance and the prevention of autoimmune diseases and are typically contemporaneously characterized by their CD3+CD4+CD25high CD127lowFOXP3+ phenotype. It is important to note that FOXP3+ Tregs exhibit substantial diversity in their origin, phenotypic characteristics, and function. Identifying reliable markers is crucial to the accurate identification, quantification, and assessment of Tregs in health and disease, as well as the enrichment and expansion of viable cells for adoptive cell therapy. In our comprehensive review, we address the contributions of various markers identified in the last two decades since the master transcriptional factor FOXP3 was identified in establishing and enriching purity, lineage stability, tissue homing and suppressive proficiency in CD4+ Tregs. Additionally, our review delves into recent breakthroughs in innovative Treg-based therapies, underscoring the significance of distinct markers in their therapeutic utilization. Understanding Treg subsets holds the key to effectively harnessing human Tregs for immunotherapeutic approaches

The Use of ProteoTuner Technology to Study Nuclear Factor κB Activation by Heavy Ions

Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects

Effects of space-relevant radiation on pre-osteoblasts

Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ~150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may provide the necessary foundation for the development for space travel countermeasures

The impact of mask use on social categorization

Here we examined whether one’s perceptual style in viewing own- and other-race faces is associated with performance and bias in social categorization by race, and whether mask use modulates the perceptual style and social categorization effects. We found that Asian participants who adopted more eyes-focused eye movement patterns when viewing Asian faces had a larger bias to judge 50% Asian-Caucasian face morphs as Asian. However, although mask use made participants’ viewing pattern more eyes-focused, it did not change this bias in judging morphed faces, or other-race advantage in social categorization speed. These results suggest that information from the eye region may be sufficient to induce these social categorization effects, and that transient perceptual input change due to mask use does not modulate these social categorization effects. Thus, effects and biases in social categorization may be impervious to mask use. These findings have important implications for social interaction during the pandemic

Studying the effect of self-selected background music on reading task with eye movements

Abstract Using background music (BGM) during learning is a common behavior, yet whether BGM can facilitate or hinder learning remains inconclusive and the underlying mechanism is largely an open question. This study aims to elucidate the effect of self-selected BGM on reading task for learners with different characteristics. Particularly, learners’ reading task performance, metacognition, and eye movements were examined, in relation to their personal traits including language proficiency, working memory capacity, music experience and personality. Data were collected from a between-subject experiment with 100 non-native English speakers who were randomly assigned into two groups. Those in the experimental group read English passages with music of their own choice played in the background, while those in the control group performed the same task in silence. Results showed no salient differences on passage comprehension accuracy or metacognition between the two groups. Comparisons on fine-grained eye movement measures reveal that BGM imposed heavier cognitive load on post-lexical processes but not on lexical processes. It was also revealed that students with higher English proficiency level or more frequent BGM usage in daily self-learning/reading experienced less cognitive load when reading with their BGM, whereas students with higher working memory capacity (WMC) invested more mental effort than those with lower WMC in the BGM condition. These findings further scientific understanding of how BGM interacts with cognitive tasks in the foreground, and provide practical guidance for learners and learning environment designers on making the most of BGM for instruction and learning

Involvement of Runx2 in the differentiation process of osteoblastic precursor cells after radiation exposure

Astronauts on exploratory space missions will experience a complex environment that includes microgravity and radiation. While the deleterious effects of unloading on bone are well established, fewer studies have focused on the effects of radiation. Space radiation produces distinct biological damages which, up to now, little is known about the correlation between radiation exposure and bone tissue. In our study we used osteoblastic precursor cells to investigate the radiation response of bone cells. Effects of radiation on differentiation were investigated by their ability to deposit extracellular matrices that mineralize under in vitro culture conditions using the histochemical Alizarin Red Staining (ARS). Calcium precipitation was detected in a bright red color already ten days after exposure to X-rays for doses up to 10 Gy. Notably, our results indicate that exposure to higher radiation doses could be correlated to a pronounced staining of the extracellular matrix. In order to gain more detailed insights into the osteoblast specific mineralization process, the transcriptional expression level of Runx2 was analysed. Our studies suggest that space relevant radiation significantly modulates the mineralization process and effectively modulates the gene expression levels of Runx2 involved in the differentiation of osteoblasts. In conclusion, the presented data allow the suggestion that exposure to ionizing radiation interferes with bone formation at the level of cellular differentiation

DNA-damage, survival, differentiation, and matrix mineralization in vitro of a murine multipotent mesenchymal precursor cell line

Radiation therapy is one of the most effective and indispensable treatment modalities for cancer patients. Known tissue complications caused by radiation-induced stem cell depletion, may result in structural and functional alterations of the surrounding matrix. Although, studies have demonstrated that ionizing radiation can induce apoptosis and senescence, little is known about the effects of therapeutic irradiation concerning the commitment of mesenchymal stem cells to the osteoblastic lineage. C3H10T1/2 clone eight cells were used reflecting an early stage of differentiation. Notably, radiation doses of 2 Gy reduced proliferation, but had no significant effect on cell viability. Cell cycle analysis revealed that the yield of cells captured in the G₂/M phase of the cell cycle was markedly and dose-dependently increased. Instead of apoptosis we detected increased activity of stress-induced premature cellular senescence. Histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix revealed positive staining for alizarin red S. Expression of TP53 encoding for tumour suppressor protein p53 and its downstream target cyclin-dependent kinase inhibitor 1A (p21(Cip1/Waf1)) were significantly increased. Gene expression analysis of two osteoblast specific genes, Runx2 and osteocalcin were assessed. Here, we confirmed that exposure to X-rays was dose dependently effective in decreasing cellular survival. Our results indicate that the direct impairment of proliferation and osteogenic differentiation potential of MSCs by irradiation may contribute partly to post-irradiation osteoporosis

Estimating Abundance of Siberian Roe Deer Using Fecal-DNA Capture-Mark-Recapture in Northeast China

It is necessary to estimate the population abundance of deer for managing their populations. However, most estimates are from high-density populations inhabiting the forests of North America or Europe; there is currently a lack of necessary knowledge regarding low-density deer populations in different forest habitats. In this article, we used fecal DNA based on the capture-mark-recapture method to estimate the population abundance of Siberian roe deer (Capreolus pygargus) in Liangshui National Nature Reserve in the Lesser Xing’an Mountains, northeast China, where the deer population was found to be of a low density by limited studies. We used a robust survey design to collect 422 fecal pellet groups in 2016 and extracted DNA from those samples, generating 265 different genotypes; we thus identified 77 deer individuals based on six microsatellite markers (Roe1, Roe8, Roe9, BM757, MB25 and OarFCB304). With capture and recapture records of these 77 individuals, the abundance of roe deer was estimated to be 87 deer (80–112, 95% CI) using the Program CAPTURE. Using an effective sampling area which resulted from the mean maximum recapture distance (MMRD), we converted the population abundance to a density of 2.9 deer/km2 (2.7–3.7, 95% CI). Our study estimated the roe deer population abundance by a feces-based capture-mark-recapture approach in northeast China, successfully demonstrating the applicability of non-invasive genetic sampling in monitoring populations of deer in this area, which contributes to the development of low-density deer population ecology and management

X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of β-glycerophosphate (β-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor β1 (TGF-β1), osteocalcin (OCN), and p21 (CDKN1A) was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-β1, OCN, and CDKN1A (p21) gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24 h after irradiation at 2 and 4 Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells

Cell cycle delay in murine pre-osteoblasts is more pronounced after exposure to high-LET compared to low-LET radiation

Space radiation contains a complex mixture of particles comprised primarily of protons and high-energy heavy ions. Radiation risk is considered one of the major health risks for astronauts who embark on both orbital and interplanetary space missions. Ionizing radiation dosedependently kills cells, damages genetic material, and disturbs cell differentiation and function. The immediate response to ionizing radiation-induced DNA damage is stimulation of DNA repair machinery and activation of cell cycle regulatory checkpoints. To date, little is known about cell cycle regulation after exposure to space-relevant radiation, especially regarding bone-forming osteoblasts. Here, we assessed cell cycle regulation in the osteoblastic cell line OCT-1 after exposure to various types of space-relevant radiation. The relative biological effectiveness (RBE) of ionizing radiation was investigated regarding the biological endpoint of cellular survival ability. Cell cycle progression was examined following radiation exposure resulting in different RBE values calculated for a cellular survival level of 1 %. Our findings indicate that radiation with a linear energy transfer (LET) of 150 keV/μm was most effective in inducing reproductive cell killing by causing cell cycle arrest. Expression analyses indicated that cells exposed to ionizing radiation exhibited significantly up-regulated p21(CDKN1A) gene expression. In conclusion, our findings suggest that cell cycle regulation is more sensitive to high- LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression