Dynamics of pathomorphological changes of ultrastructural organization of intervertebral disc at different terms of experimental opioid influence and with its cancellation

Background. Patients with degenerative diseases of the spine - "difficult patients". Often, exhausted by long-term pain, they have a dependence, and sometimes tolerance to various analgesics, which greatly reduces the effectiveness of routinely used in the hospital anesthesia. Objective. The aim of our work was to study the features of pathomorphological manifestations of the structural components of the intervertebral disc at different terms of opioid influence and at the difference at the ultrastructural level in the experiment. Methods. The material of the study were sexually mature, white, nonlinear rats - males in the amount of 90 hundred animals, weighing 92 - 103 g, aged 4.5 months. Animals were injected with Nalbuphine at home, once daily for one day (10-11 am) for 42 days. The initial dose of Nalbuphine was 8 mg / kg during the first week, 15 mg / kg during the second week; 20 mg / kg during the third week; 25 mg / kg during the fourth week; 30 mg / kg during the fifth week and 35 mg / kg during the sixth week of the experimental opioid effect. Thus created the conditions for chronic opioid exposure. Intervertebral discs of rats were used as material for ultrastructural study. Results and conclusion. As a result of our study, we found that at the end of 21 days we found the progression of alternative changes in the cellular elements of the gelatinous nucleus, characterized by the development of necrotic changes in notochondral cells, as well as chondroptosis of chondrocytes. Preserved notochondral cells were rarely visualized. In some places, there were notochondral cells in which the nucleus disintegrated into separate fragments filled with heterochromatin, and the remains of organelle membranes were localized in the enlightened cytoplasm. Pronounced destructive changes were found in chondrocytes. After 28 days, the changes progressed, this was manifested by the fact that in the pulpal nucleus there were extensive cell-free zones filled with a significant amount of granular intensely osmophilic mass. Notochondral cells and most chondrocytes underwent necrotic changes. After 35 days at the ultrastructural level revealed pronounced changes in the structural elements of the gelatinous nucleus and fibrous ring. Extensive cell-free zones were localized in the gelatinous nucleus, with a somewhat compacted matrix in which an intensely osmophilic fine-grained deep mass accumulated. With the abolition of opioid exposure at the end of 56 days, we found pronounced changes in notochondral cells and in the vast majority of chondrocytes. The matrix of the gelatinous nucleus was compacted, it showed thickened collagen fibrils. Most of the cellular elements of the gelatinous nucleus were at different stages of necrosis, and some chondrocytes - chondroptosis. Vacuoles filled with enlightened contents also appeared in the cytoplasm. The nucleus was compacted, condensation of chromatin was observed. In such areas, the fibrils of collagen fibers were loose, stratified, disintegrated and lysed.

In Vitro Anti-Biofilm Activity of Hydrogen-Peroxide Generating Electrochemical Bandage Against Yeast Biofilms

Wound infections are caused by bacteria and/or fungi. The presence of fungal biofilms in wound beds presents a unique challenge, as fungal biofilms may be difficult to eradicate. The goal of this work was to assess the in vitro anti-biofilm activity of a H 2 O 2 -producing electrochemical bandage (e-bandage) against 15 yeast isolates representing commonly-encountered species. Time-dependent decreases in viable biofilm CFU counts of all isolates tested were observed, resulting in no visible colonies with 48 hours of exposure by plate culture. Fluorescence microscopic analysis showed extensive cell membrane damage of biofilm cells after e-bandage treatment. Reductions in intracellular ATP levels of yeast biofilm cells were recorded post e-bandage treatment. Our results suggest that exposure to H 2 O 2 -producing e-bandages reduce in vitro viable cell counts of yeast biofilms, making this a potential new topical treatment approach for fungal wound infections.

The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells

Background DNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESCs) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss. Results We determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1-deficient mESCs. Conclusions GSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma

Loss-of-function mutations in the RB1 tumour suppressor are key drivers in cancer, including osteosarcoma. RB1 loss-of-function compromises genome-maintenance and hence could yield vulnerability to therapeutics targeting such processes. Here we demonstrate selective hypersensitivity to clinically-approved inhibitors of Poly-ADP-Polymerase1,2 inhibitors (PARPi) in RB1-defective cancer cells, including an extended panel of osteosarcoma-derived lines. PARPi treatment results in extensive cell death in RB1-defective backgrounds and prolongs survival of mice carrying human RB1-defective osteosarcoma grafts. PARPi sensitivity is not associated with canonical homologous recombination defect (HRd) signatures that predict PARPi sensitivity in cancers with BRCA1,2 loss, but is accompanied by rapid activation of DNA replication checkpoint signalling, and active DNA replication is a prerequisite for sensitivity. Importantly, sensitivity in backgrounds with natural or engineered RB1 loss surpasses that seen in BRCA-mutated backgrounds where PARPi have established clinical benefit. Our work provides evidence that PARPi sensitivity extends beyond cancers identifiable by HRd and advocates PARP1,2 inhibition as a personalised strategy for RB1-mutated osteosarcoma and other cancers.

Critical role of H3K27 methylation in cell fate determination of two cell lineages in male gametophyte

During angiosperm male gametogenesis, microspores divide to produce a vegetative cell (VC) and a male germline (MG), each with a distinct cell fate. How the MG cell/VC fate is determined remains largely unknown. Here, we report that H3K27me3 is essential for VC fate commitment and H3K27me3 erasure contributes to the MG cell fate initiation in male gametophyte of Arabidopsis. The VC-targeted H3K27me3 erasure disturbed the VC development and resulted in the VC fate shifting towards a gamete destination, which suggests that MG cells require H3K27me3 erasure for triggering the gamete cell fate. Multi-omics and cytologic analysis confirmed the occurrence of extensive cell identity transition due to H3K27me3 erasure. Therefore, we experimentally confirm that the MG cell/VC fate is epigenetically regulated. The H3K27 methylation plays a critical role in the guidance of MG cell/VC fate determination for pollen fertility in Arabidopsis. Our work also provides new evidences for two previous hypotheses that the germline cell fate is specified by the differential distribution of yet unknown determinant, and VC maintains the microspore's default program, namely the H3K27me3 setting, whereas MG requires reprogramming.

Modulation of Inherent Niches in 3D Multicellular MSC Spheroids Reconfigures Metabolism and Enhances Therapeutic Potential

Multicellular spheroids show three-dimensional (3D) organization with extensive cell–cell and cell–extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential by tuning the cultivation parameters and thus the inherent niche of 3D MSC spheroids. Significantly increased expression of multiple pro-regenerative paracrine signaling molecules and immunomodulatory factors by MSCs was observed after optimizing the conditions for spheroid culture. Moreover, these alterations in cellular behaviors may be associated with not only the hypoxic niche developed in the spheroid core but also with the metabolic reconfiguration of MSCs. The present study provides efficient methods for manipulating the therapeutic capacity of 3D MSC spheroids, thus laying solid foundations for future development and clinical application of spheroid-based MSC therapy for regenerative medicine.

Pathway dynamics can delineate the sources of transcriptional noise in gene expression

Single-cell expression profiling opens up new vistas on cellular processes. Extensive cell-to-cell variability at the transcriptomic and proteomic level has been one of the stand-out observations. Because most experimental analyses are destructive we only have access to snapshot data of cellular states. This loss of temporal information presents significant challenges for inferring dynamics, as well as causes of cell-to-cell variability. In particular, we typically cannot separate dynamic variability from within cells ('intrinsic noise') from variability across the population ('extrinsic noise'). Here we make this non-identifiability mathematically precise, allowing us to identify new experimental set-ups that can assist in resolving this non-identifiability. We show that multiple generic reporters from the same biochemical pathways (e.g. mRNA and protein) can infer magnitudes of intrinsic and extrinsic transcriptional noise, identifying sources of heterogeneity. Stochastic simulations support our theory, and demonstrate that 'pathway-reporters' compare favourably to the well-known, but often difficult to implement, dual-reporter method.

Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster

Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. We identified Dhc64Cm115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. We propose that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development.

Effect of Radiation Emitted by Wireless Devices on Male Reproductive Hormones: A Systematic Review

Exposure to radiofrequency electromagnetic radiation (RF-EMR) from various wireless devices has increased dramatically with the advancement of technology. One of the most vulnerable organs to the RF-EMR is the testes. This is due to the fact that testicular tissues are more susceptible to oxidative stress due to a high rate of cell division and mitochondrial oxygen consumption. As a result of extensive cell proliferation, replication errors occur, resulting in DNA fragmentation in the sperm. While high oxygen consumption increases the level of oxidative phosphorylation by-products (free radicals) in the mitochondria. Furthermore, due to its inability to effectively dissipate excess heat, testes are also susceptible to thermal effects from RF-EMR exposure. As a result, people are concerned about its impact on male reproductive function. The aim of this article was to conduct a review of literature on the effects of RF-EMR emitted by wireless devices on male reproductive hormones in experimental animals and humans. According to the findings of the studies, RF-EMR emitted by mobile phones and Wi-Fi devices can cause testosterone reduction. However, the effect on gonadotrophic hormones (follicle-stimulating hormone and luteinizing hormone) is inconclusive. These findings were influenced by several factors, which can influence energy absorption and the biological effect of RF-EMR. The effect of RF-EMR in the majority of animal and human studies appeared to be related to the duration of mobile phone use. Thus, limiting the use of wireless devices is recommended.

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as identified by sCRilecs-seq

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the β-lactam antibiotic amoxicillin, which is the first intention therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin- resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. We show that this phenotype is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that septal peptidoglycan synthesis is more sensitive to reduced Und-P levels than peripheral peptidoglycan synthesis. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.