Investigations concerning the impact of consumption of hot beverages on acute cytotoxic and genotoxic effects in oral mucosa cells

Consumption of very hot beverages and foods increases the incidence of oral and esophageal cancer but the mechanisms are not known and the critical temperature is not well defined. We realized a study with exfoliated cells from the oral cavity of individuals (n = 73) that live in an area in Iran which has the highest incidence of EC worldwide. Consumption of beverages at very high temperatures is a characteristic feature of this population. We analyzed biomarkers which are (i) indicative for genetic instability (micronuclei that are formed as a consequence of chromosomal damage, nuclear buds which are a consequence of gene amplifications and binucleated cells which reflect mitotic disturbances), (ii) markers that reflect cytotoxic effects (condensed chromatin, karyorrhectic, karyolitic and pyknotic cells), (iii) furthermore, we determined the number of basal cells which is indicative for the regenerative capacity of the buccal mucosa. The impact of the drinking temperature on the frequencies of these parameters was monitored with thermometers. We found no evidence for induction of genetic damage but an increase of the cytotoxic effects with the temperature was evident. This effect was paralleled by an increase of the cell division rate of the mucosa which was observed when the temperature exceeded 60 °C. Our findings indicate that cancer in the upper digestive tract in drinkers of very hot beverages is not caused by damage of the genetic material but by an increase of the cell division rate as a consequence of cytotoxic effects which take place at temperatures over 60 °C. It is known from earlier experiments with rodents that increased cell divisions lead to tumor promotion in the esophagus. Our findings provide a mechanistic explanation and indicate that increased cancer risks can be expected when the drinking temperature of beverages exceeds 60 °C.

Long-lasting and responsive DNA/enzyme-based programs in serum-supplemented extracellular media

DNA molecular programs are emerging as promising pharmaceutical approaches due to their versatility for biomolecular sensing and actuation. However, the implementation of DNA programs has been mainly limited to serum-deprived in vitro assays due to the fast deterioration of the DNA reaction networks by the nucleases present in the serum. Here, we show that DNA/enzyme programs are functional in serum for 24h but are latter disrupted by nucleases that give rise to parasitic amplification. To overcome this, we implement 3-letter code networks that suppress autocatalytic parasites while still conserving the functionality of DNA/enzyme programs for at least 3 days in the presence of 10% serum. In addition, we define a new buffer that further increases the biocompatibility and conserves responsiveness to changes in molecular composition across time. Finally, we demonstrate how serum-supplemented extracellular DNA molecular programs remain responsive to molecular inputs in the presence of living cells, having responses 6-fold faster than cellular division rate and are sustainable for at least 3 cellular divisions. This demonstrates the possibility of implementing in situ biomolecular characterization tools for serum-demanding in vitro models. We foresee that the coupling of chemical reactivity to our DNA programs by aptamers or oligonucleotide conjugations will allow the implementation of extracellular synthetic biology tools, which will offer new biomolecular pharmaceutical approaches and the emergence of complex and autonomous in vitro models.

Adhesion strength between cells regulate non-monotonic growth by a biomechanical feedback mechanism

We probe the interplay between intercellular interactions and pressure fluctuations associated with single cells in regulating cell proliferation using simulations of a minimal model for three-dimensional multicellular spheroid (MCS) growth. The emergent spatial variations in the cell division rate, that depends on the location of the cells within the MCS, is regulated by intercellular adhesion strength (f^{ad}). This in turn results in non-monotonic proliferation of cells in the MCS with varying adhesion strength, which accords well with experimental results. A biomechanical feedback mechanism coupling the f^{ad} and cell-dependent pressure fluctuations relative to a threshold value (p_c) determines the onset of a dormant phase, and explains the non-monotonic proliferation response. Increasing f^{ad} from low values enhances cell proliferation because pressure on individual cells is smaller compared to p_c. In contrast, at high f^{ad}, cells readily become dormant and cannot rearrange effectively, leading to arrested cell proliferation. Our work, which shows that proliferation is regulated by pressure-adhesion feedback loop, may be a general feature of tumor growth.

A Small Fraction of Progenitors Differentiate Into Mature Adipocytes by Escaping the Constraints on the Cell Structure

Differentiating 3T3-L1 pre-adipocytes are a mixture of non-identical culture cells. It is vital to identify the cell types that respond to the induction stimulus to understand the pre-adipocyte potential and the mature adipocyte behavior. To test this hypothesis, we deconvoluted the gene expression profiles of the cell culture of MDI-induced 3T3-L1 cells. Then we estimated the fractions of the sub-populations and their changes in time. We characterized the sub-populations based on their specific expression profiles. Initial cell cultures comprised three distinct phenotypes. A small fraction of the starting cells responded to the induction and developed into mature adipocytes. Unresponsive cells were probably under structural constraints or were committed to differentiating into alternative phenotypes. Using the same population gene markers, similar proportions were found in induced human primary adipocyte cell cultures. The three sub-populations had diverse responses to treatment with various drugs and compounds. Only the response of the maturating sub-population resembled that estimated from the profiles of the mixture. We then showed that even at a low division rate, a small fraction of cells could increase its share in a dynamic two-populations model. Finally, we used a cell cycle expression index to validate that model. To sum, pre-adipocytes are a mixture of different cells of which a limited fraction become mature adipocytes.

Inhibition of neutrophilic serine proteases: a new direction of pathogenetic therapy in patients with psoriasis

Псориаз является хроническим воспалительным иммуно-опосредованным заболеванием, характеризующимся повышенной скоростью деления кератиноцитов и нарушением их дифференцировки. Несмотря на стремительную разработку и внедрение новых системных лекарственных средств, наружная терапия остается неотъемлемой частью лечения больных псориазом. Основным её недостатком является низкая избирательность и неспецифичность противовоспалительного действия при различных дерматозах, в том числе и псориазе, что может проявляться как развитием нежелательных явлений, так и недостаточной терапевтической эффективностью. В данном обзоре представлена концепция нового направления терапии больных псориазом на основании разработки препарата, блокирующего сериновые протеазы. Приведены сведения о современных механизмах развития псориаза и роли ИЛ-36-опосредованного воспаления. Освещены вопросы о сериновых протеазах кожи, а также их эндогенных и синтезированных низкомолекулярных ингибиторах. Показаны перспективы реализации данного направления, в том числе результаты доклинических исследований. Дальнейшее развитие этого направления позволит усовершенствовать подходы к лечению как ограниченных, так и распространенных форм заболевания, что может принципиально поменять подходы ведения больных псориазом. Psoriasis is a chronic inflammatory immune-mediated disease characterized by increased division rate and impaired differentiation of keratinocytes. Despite the rapid development and introduction of new systemic drugs, external therapy remains an integral part of the treatment of patients with psoriasis. A major disadvantage of this treatment is the low selectivity and the non-specificity of its anti-inflammatory action in various dermatoses, including psoriasis, which may manifest itself as adverse effects and insufficient therapeutic efficacy. This review presents a new direction in the treatment of psoriasis based on development of a drug that would inhibit serine proteases. Current information about mechanisms of psoriasis and the role of IL-36-mediated inflammation is presented. Also, the review addresses serine proteases of the skin, specifically their endogenous and synthetic low-molecular inhibitors. Prospects for implementation of this novel treatment, including results of preclinical studies, are described. Further development of this direction will allow improving the treatment of both limited and widespread forms of psoriasis, which may fundamentally change the approach to managing patients with this disease.

Synthesis of Epitope-Targeting Antibody Based on Native Protein–Protein Interactions for Ftsz Filamentation Suppressor

Phage display and biopanning is a powerful tool for generating binding molecules for a specific target. However, the selection process based on binding affinity provides no assurance for the antibody’s affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein–protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the antibody, and a nonrelated CDR loop (in the grafted antibody) was randomized by phage display techniques. In this construction of antibodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence weakly functionalized the antibody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein–protein interactions. Antibody fragments with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) were constructed and completely inhibited the polymerization of FtsZ. Consequently, the expression of these fragments drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein–protein interactions for steady epitope-specific evolutionary engineering.

Hyperosmotic Shock Transiently Accelerates Constriction Rate in Escherichia coli

Bacterial cells in their natural environments encounter rapid and large changes in external osmolality. For instance, enteric bacteria such as Escherichia coli experience a rapid decrease when they exit from host intestines. Changes in osmolality alter the mechanical load on the cell envelope, and previous studies have shown that large osmotic shocks can slow down bacterial growth and impact cytoplasmic diffusion. However, it remains unclear how cells maintain envelope integrity and regulate envelope synthesis in response to osmotic shocks. In this study, we developed an agarose pad-based protocol to assay envelope stiffness by measuring population-averaged cell length before and after a hyperosmotic shock. Pad-based measurements exhibited an apparently larger length change compared with single-cell dynamics in a microfluidic device, which we found was quantitatively explained by a transient increase in division rate after the shock. Inhibiting cell division led to consistent measurements between agarose pad-based and microfluidic measurements. Directly after hyperosmotic shock, FtsZ concentration and Z-ring intensity increased, and the rate of septum constriction increased. These findings establish an agarose pad-based protocol for quantifying cell envelope stiffness, and demonstrate that mechanical perturbations can have profound effects on bacterial physiology.

Genetic polymorphism of ToxB+ Pyrenophora tritici-repentis strains

BACKGROUND: The phytotoxin Ptr ToxB as well as Ptr ToxA is one of the pathogenic factors of Pyrenophora tritici-repentis, that cause leaf chlorosis on susceptible wheat varieties, and is encoded by ToxB gene. P. tritici-repentis strains with ToxB gene are rather rare worldwide. MATERIALS AND METHODS: The object of the study was 37 strains isolated from the leaves of wheat grown in Greece. The virulence of the strains was analyzed and the presence of effector genes as well as the average copy number of ToxB was determined. RESULTS: The race composition of P. tritici-repentis population turned out to be mainly represented by the avirulent race 4 (50% of the strains). Strains of race 1 were not found, while strains of other races were found with a low frequency in the population. All analyzed P. tritici-repentis strains had ToxB gene in the genome, while its homologues and ToxA gene were not detected. The average copy number (R) of ToxB in three P. tritici-repentis strains varied from 0.24 to 1.22. The average copy number of ToxB in the mitotic generation of P. tritici-repentis Gr8 strain, which was characterized by the lowest value of R = 0.24, varied from 0.01 to 0.74 and, on average, turned out to be 2 times higher than in the original strain Gr8. CONCLUSION: Presumably, P. tritici-repentis has a mechanism that gives ToxB+ nuclei an advantage in the division rate over ToxB nuclei. This mechanism indicates the existence of an additional function of this gene that is not associated with pathogenicity.

3′,4′-Dihydroxyflavonol Modulates the Cell Cycle in Cancer Cells: Implication as a Potential Combination Drug in Osteosarcoma

New agents are demanded to increase the therapeutic options for osteosarcoma (OS). Although OS is the most common bone cancer in children and adolescents, it is considered a rare disorder. Therefore, finding adjuvant drugs has potential to advance therapy for this disease. In this study, 3′,4′-dihydroxyflavonol (DiOHF) was investigated to assess the effects in OS cellular models in combination with doxorubicin (Dox). MG-63 and U2OS human OS cells were exposed to DiOHF and Dox and tested for cell viability and growth. To elucidate the inhibitory effects of DiOHF, additional studies were conducted to assess apoptosis and cell cycle distribution, gene expression quantification of cell cycle regulators, and cytokinesis-block cytome assay to determine nuclear division rate. DiOHF decreased OS cell growth and viability in a concentration-dependent manner. Its combination with Dox enabled Dox dose reduction in both cell lines, with synergistic interactions in U2OS cells. Although no significant apoptotic effects were detected at low concentrations, cytostatic effects were demonstrated in both cell lines. Incubation with DiOHF altered cell cycle dynamics and resulted in differential cyclin and cyclin-dependent kinase expression. Overall, this study presents an antiproliferative action of DiOHF in OS combination therapy via modulation of the cell cycle and nuclear division.

P–223 The necrotic oocyte: does the uncontrolled release of cell contents affect adjacently, group cultured embryos?

Study question Is embryo utilisation rate, embryo morphokinetics and the incidence of irregular divisions affected when embryos are group-cultured adjacent to a necrotic oocyte? Summary answer This study demonstrates that embryos cultured adjacent to necrotic oocytes appear to be unaffected both in terms of utilisation, morphokinetics and incidence of irregular divisions. What is known already Necrosis is a form of uncontrolled cell death, usually resulting from external injury, causing the cell’s contents to release into the surrounding environment1. A cell undergoing necrosis will first visibly swell before the collapse of the plasma membrane causes it to subsequently shrink and the cell to lyse2. An escalation of inflammation occurs due to the release of intracellular factors3. Neighbouring embryos are believed to be negatively affected by a necrotic oocyte with some laboratories choosing to remove necrotic oocytes from culture dishes, however, little is known regarding this impact. Study design, size, duration The project was a single site, retrospective cohort analysis using time-lapse data from August 2017 to December 2018. Only patients with at least one necrotic oocyte, a minimum of one adjacent embryo to the necrotic oocyte and those cultured in the EmbryoScope+® were included in the analysis. Participants/materials, setting, methods The study included 868 embryos from 89 patients. The embryos were categorised as adjacent to a necrotic oocyte (group 1, n = 208) and not adjacent to a necrotic oocyte (group 2, n = 660). The utilisation rate and irregular division rate were analysed using a Chi-squared test, the morphokinetic parameters was analysed using a t-test. Morphokinetic data included; tPB2, tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, t9, tSC, tM, tSB and tB. Main results and the role of chance Utilisation rate between the two groups was not significantly different (group 1; 40.9% versus group 2; 47.6%, p = 0.09). Incidence of irregular division was not significantly different between the two groups (group 1; 24.0% vs group 2; 21.7%, p = 0.51). No morphokinetic parameter was statistically significantly different when comparing group 1 to group 2, respectively: tPB2, 3.61 vs 3.73, p = 0.38; tPNa, 7.01 vs 6.91, p = 0.59; tPNf, 23.64 vs 23.66, p = 0.95; t2, 3.44 vs 2.98, p = 0.09; t3, 14.56 vs 14.41, p = 0.75; t4, 15.96 vs 15.8, p = 0.77; t5, 15.96 vs 15.8, p = 0.77; t6, 30.33 vs 30.46, p = 0.86; t7, 33.11 vs 33.16, p = 0.95; t8, 37.93 vs 36.92, p = 0.34; t9, 48.66 vs 48.97, p = 0.73; tSC, 58.04 vs 57.89, p = 0.88; tM, 74.02 vs 73.76, p = 0.8; tSB, 75.55 vs 75.42, p = 0.9; tB, 87.06 vs 87.2, p = 0.91. Limitations, reasons for caution The time at which the oocytes became necrotic was not analysed therefore the effect, if any, of exposure time could not be determined. Of the 169 necrotic oocytes, two were from IVF and 167 from ICSI; the increased exposure of the embryos derived from ICSI was not controlled for. Wider implications of the findings: Necrotic oocytes are easily identified in standard culture observations and in time-lapse imaging, therefore, their removal may be an unnecessary practice. More harm could be caused by removing the dish from the incubator, as this would unnecessarily expose any viable embryos contained within the dish to a suboptimal environment. Trial registration number Not applicable