Changes In Ultrastructure Of Cells Research Articles

Lead-induced changes in plant cell ultrastructure: an overview.

Lead (Pb) is one of the most harmful toxic metals and causes severe damage to plants even at low concentrations. Pb inhibits plant development, reduces photosynthesis rates, and causes metabolic disfunctions. Plant cells display these alterations in the form of abnormal morphological modifications resulting from ultrastructural changes in the cell wall, plasma membrane, chloroplast, endoplasmic reticulum, mitochondria, and nuclei. Depending on plant tolerance capacity, the ultrastructural changes could be either a sign of toxicity that limits plant development or an adaptive strategy to cope with Pb stress. This paper gathers data on Pb-induced changes in cell ultrastructure observed in many tolerant and hyperaccumulator plants and describes the ultrastructural changes that appear to be mechanisms to alleviate Pb toxicity. The different modifications caused by Pb in cell organelles are summarized and reinforced with hypotheses that provide an overview of plant responses to Pb stress and explain the physiological and morphological changes that occur in tolerant plants. These ultrastructural modifications could help assess the potential of plants for use in phytoremediation.

Effect of a thiohydantoin salt derived from l-Arginine on Leishmania amazonensis and infected cells: Insights from biological effects to molecular docking interactions

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor.

In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

Significance of erythropoesis correction in preoperative preparation in the surgical treatment of patients with colorectal cancer

Background. Anemic syndrome, which accompanies the development of pathology in patients with colorectal cancer, can cause serious early postoperative complications. The question of how preoperative preparation activities influence the state of the intestinal structures and the clinical situation in general is relevant. Of particular importance in this situation is the correction of anemic syndrome before surgery, using hematopoietic stimulants. Objective. The purpose of the study is to analyze the dynamics of erythropoiesis and changes in tissue and cell ultrastructure in intraoperative colon biopsy specimens after stimulation of erythropoiesis in preoperative preparation in the surgical treatment of patients with colorectal cancer. Methods. The study of red bone marrow and the wall of the large intestine using transmission electron microscopy. Results. Most patients with colorectal cancer are characterized by the development of signs of ineffective erythropoiesis with a moderate slowdown in the differentiation of erythroblasts and a delay in the release of mature erythrocytes into the systemic circulation. Stimulation of erythropoiesis with the help of epoetin leads to an increase in the functional activity of the central macrophages of the erythropoietic islets of the red bone marrow and the enhancement of their intercellular association with maturing erythroid cells. This is accompanied by the formation of a large number of full-fledged erythrocytes, an increase in the numerical density of cells of the erythroid series and the normalization of their ultrastructure. Conclusion. Stimulation of erythropoiesis with the help of epoetin in the preoperative period causes the inhibition of endothelial dysfunction due to the restriction of dystrophic and destructive changes in endothelial cell organelles, stabilization of their membranes, prevention of arteriole spasm, erythrocyte stasis and erythrocyte phenomena. As a result, the phenomena of infiltration, perivascular and interstitial edema in the composition of the membranes of the intestinal wall are much less pronounced than in the group of patients without preoperative activation of erythropoiesis.

A novel metabolite of Streptomyces coeruleorubidus exhibits antibacterial activity against Streptococcus agalactiae through modulation of physiological performance, inflammatory cytokines, apoptosis, and oxidative stress-correlated gene expressions in Nile tilapia (Oreochromis niloticus)

Using the unique structures found in natural materials to produce new antibacterial drugs is crucial. Actinobacteria is well-known for its ability to produce naturally occurring chemicals with a variety of structural features that can be used as weapons against infectious bacteria. In the present study, the Streptomyces coeruleorubidus metabolites were characterized and their efficacy in suppressing Streptococcus agalactiae growth was carried out both in vitro and in vivo. The metabolites of S. coeruleorubidus were purified and identified as octasiloxane-hexadecamethyl (OHM). In vivo antibacterial activity of OHM revealed an inhibitory minimum concentration value of 0.5 μg/ml against S. agalactiae and induced ultrastructural cell changes revealed by scanning electron microscope. The safe concentration of OHM was determined as 0.8 mg/L for Nile tilapia. Four in vivo treatments were treated with 0 and 0.8 mg/L OHM and with or without challenge by S. agalactiae (1 × 107 CFU/mL) named control, OHM, S. agalactiae, and S. agalactiae + OHM groups. The OHM treatment improved the survival of Nile tilapia by 33.33% than S. agalactiae challenge group. Waterborne OHM treatment significantly mitigated the deleterious effects of S. agalactiae on hematological, hepato-renal functions, stress indicators, and antioxidant balance. OHM significantly alleviated nitric oxide levels, complement 3, IgM, and lysozyme activity, downregulation of liver antioxidant genes expression in S. agalactiae group. Furthermore, the addition of OHM to challenged fish with S. agalactiae-significantly reversed dramatic negative regulation of inflammatory, apoptosis, and immune related gene expression (caspase-3, bax, pcna, tnf-α, ifn-γ, il-8 il-1β, il-10, tgf-β, and bcl-2 in the Nile tilapia spleen. Additionally, the damaged hepatic and splenic structure induced by bacterial infection was restored with OHM treatment. Finally, S. coeruleorubidus metabolites (mainly OHM) revealed in vitro and in vivo antibacterial activity and showed alleviated effects on the physiological status of S. agalactiae infected tilapia.

Uptake, accumulation and toxicity of short chain chlorinated paraffins to wheat (Triticum aestivum L.)

Short chain chlorinated paraffins (SCCPs) are ubiquitous persistent organic pollutants. They have been widely detected in plant-based foods and might cause adverse impacts on humans. Nevertheless, uptake and accumulation mechanisms of SCCPs in plants remain unclear. In this study, the soil culture data indicated that SCCPs were strongly absorbed by roots (root concentration factor, RCF>1) yet limited translocated to shoots (translocation factor<1). The uptake mechanism was explored by hydroponic exposure, showing that hydrophobicity and molecular size influenced the root uptake and translocation of SCCPs. RCFs were significantly correlated with logKow values and molecular weights in a parabolic curve relationship. Besides, it was extremely difficult for SCCPs to translocate from shoots back to roots via phloem. An active energy-dependent process was proposed to be involved in the root uptake of SCCPs, which was supported by the uptake inhibition by the low temperature and metabolic inhibitor. Though SCCPs at environmentally relevant concentrations had no negative impacts on root morphology and chlorophyll contents, it caused obvious changes in cellular ultrastructure of root tip cells and induced a significant increase in superoxide dismutase activity. This information may be beneficial to moderate crop contamination by SCCPs, and to remedy soils polluted by SCCPs with plants.

Endoplasmic Reticulum Stress and Autophagy Are Involved in Hepatotoxicity Induced by Tributyltin.

Tributyltin (TBT), a common contaminant in aquatic ecosystems, has severe toxic effects on multiple tissues and organs, especially the liver. Previous toxicogenomic analysis has indicated that the main mechanism of TBT-induced hepatotoxicity is related to the activation of the apoptotic pathway. However, the mechanism of action occurring before the activation of apoptosis is still unclear. Herein, we applied proteomic technology to explore the protein expression profile of TBT-treated HL7702 normal human liver cells. The ultrastructural changes in cells were observed by transmission electron microscopy. After low dose (2 μΜ) TBT treatment, activation of the unfolded protein response and endoplasmic reticulum stress were observed; the expression levels of PERK, ATF6, BiP, and CHOP were significantly elevated, and splicing of XBP1 mRNA was initiated. When the TBT concentration increased to 4 μΜ, the protein levels of Beclin1, Atg3, Atg5, Atg7, and Atg12-Atg5 were significantly elevated, and the protein level of LC3Ⅰ decreased while that of LC3Ⅱ increased, suggesting the activation of autophagy. As the TBT concentration continued to increase, autophagy could not eliminate the damage, and apoptosis eventually occurred. These results indicate novel pathways of hepatotoxicity induced by TBT and provide insights for future studies.

IL-8 Links NF-κB and Wnt/β-Catenin Pathways in Persistent Inflammatory Response Induced by Chronic &lt;i&gt;Helicobacter pylori&lt;/i&gt; Infection

Helicobacter pylori (H. pylori) infection can cause persistent inflammatory response in human gastric mucosal epithelial cells, which may result in the occurrence of cancer. However, the underlying mechanism of carcinogenesis has not been elucidated yet. Herein, we established the models of chronic H. pylori infection in GES-1 cells and C57BL/6J mice. Interleukin 8 (IL-8) level was detected by ELISA. The expression of NF-κB p65, IL-8, Wnt2 and β-catenin mRNA and proteins was evaluated by real-time PCR, Western blotting, immunofluorescence staining, and immunohistochemistry. The infection of H. pylori in mice was evaluated by rapid urease test, HE staining and Warthin‒Starry silver staining. The morphological changes of gastric mucosa were observed by electron microscopy. Our results showed that in H. pylori infected gastric mucosal cells along with activation of NF-κB signaling pathway and increase of IL-8 level, the expression of Wnt2 was also increased significantly, which preliminarily indicates that IL-8 can positively regulate the expression of Wnt2. Studies in chronic H. pylori infected C57BL/6J mice models showed that there was an increased incidence of premalignant lesions in the gastric mucosa tissue. Through comparing changes of gastric mucosal cell ultrastructure and analyzing the relationship between NF-κB signaling pathway and Wnt2 expression, we found that H. pylori infection activated NF-κB signal pathways, and the massive release of IL-8 was positively correlated with the high expression of Wnt2 protein. Subsequently, the activated Wnt/β-catenin signal pathways may be involved in the malignant transformation of gastric mucosal cells. Collectively, H. pylori chronic infection may continuously lead to persistent inflammatory response: activate NF-κB pathway, promote IL-8 release and thereby activate Wnt/β-catenin pathway. IL-8 probably plays an important role of a linker in coupling these two signal pathways.

Characteristics of a New Halotolerant Arctic Strain of Carotenogenic Microalga &lt;i&gt;Halochlorella rubescens&lt;/i&gt; NAMSU SBB-20

Green microalgae capable of accumulating secondary carotenoids are the most important objects of biotechnology, and the search for new strains with unique properties, in particular, those adapted to growth at low temperatures and high salinity in the environment, is an urgent task. The NAMSU SBB-20 microalga strain was isolated from an algal-bacterial biofilm found on the coast of the White Sea in the littoral zone of the Solovetsky Archipelago. Identification of the strain showed its belonging to the species Halochlorella rubescens P.J.L.Dangeard. The species H. rubescens was first described for the White Sea. Under conditions of high light intensity, ultrastructural changes in cells are shown, among which destruction of the photosynthetic apparatus and the formation of cytoplasmic and chloroplast lipid inclusions are noted. It was shown that the culture of the NAMSU SBB-20 strain is capable of acquiring an orange color under unfavorable growth conditions. An assessment was made of the effect of the composition of the medium and the intensity of illumination on the pigment composition of the algae. The highest absolute values of the accumulation of carotenoids were noted during cultivation in light with an intensity of 150 mol PAR quanta/m2/s on BG-11 media containing no source of phosphorus (15.66 0.18 mg/L) or nitrogen (15.95 0.56 mg/L). The described strain has a biotechnological potential due to the initial halotolerance and the accumulation of high values of secondary carotenoids in the biomass.

Xue-Jie-San restricts ferroptosis in Crohn's disease via inhibiting FGL1/NF-κB/STAT3 positive feedback loop.

Crohn's disease (CD) is an incurable inflammatory bowel disease due to unclear etiology and pathogenesis. Accumulating evidences have shown the harmful role of ferroptosis in CD onset and development. Additionally, fibrinogen-like protein 1 (FGL1) has been verified to be a potential therapeutic target of CD. Xue-Jie-San (XJS) is an effective prescription for treating CD. However, its therapeutic mechanism has not been fully elucidated. This study aimed to determine whether XJS alleviating CD via regulating ferroptosis and FGL1 expression. A colitis rat model was induced by 2,4,6-trinitrobenzene sulfonic acid and treated with XJS. The disease activity indices of the colitis rats were scored. Histopathological damage was assessed using HE staining. ELISA was performed to examine inflammatory cytokines. Transmission electron microscopy was utilized to observe ultrastructure changes in intestinal epithelial cells (IECs). Iron load was evaluated by examining iron concentrations, the expressions of FPN, FTH and FTL. Lipid peroxidation was investigated through detecting the levels of ROS, 4-HNE, MDA and PTGS2. Furthermore, the SLC7A11/GSH/GPX4 antioxidant system and FGL1/NF-κB/STAT3 signaling pathway were examined. The results showed that colitis was dramatically ameliorated in the XJS-treated rats as evidenced by relief of clinical symptoms and histopathological damages, downregulation of pro-inflammatory cytokines IL-6, IL-17 and TNF-α, and upregulation of anti-inflammatory cytokine IL-10. Furthermore, XJS administration led to ferroptosis inhibition in IECs by reducing iron overload and lipid peroxidation. Mechanistically, XJS enhanced the SLC7A11/GSH/GPX4 antioxidant system negatively regulated by the FGL1/NF-κB/STAT3 positive feedback loop. In conclusion, XJS might restrain ferroptosis in IECs to ameliorate experimental colitis by inhibition of FGL1/NF-κB/STAT3 positive feedback loop.

Effect of Viral Infection on the Ultrastructural Organization of Black Currant Leaf Tissue Cells

Effect of Viral Infection on the Ultrastructural Organization of Black Currant Leaf Tissue Cells

Nano-bio interactions of upconversion nanoparticles at subcellular level: biodistribution and cytotoxicity.

Background: Modern medicine requires intensive research to find new diagnostic and therapeutic solutions. Recently, upconverting nanoparticles (UCNPs) doped with lanthanide ions have attracted significant attention. Methods: The efficient internalization of UCNPs by cells was confirmed, and their precise cellular localization was determined by electron microscopy and confocal studies. Results: UCNPs colocalized only with specific organelles, such as early endosomes, late endosomes and lysosomes. Furthermore, experiments with chemical inhibitors confirmed the involvement of endocytosis in UCNPsinternalization and helped select several mechanisms involved in internalization. Exposure to selected UCNPsconcentrations did not show significant cytotoxicity, induction of oxidative stress or ultrastructural changes in cells. Conclusion: This study suggests that UCNPs offer new diagnostic options for biomedical infrared imaging.

IL-8 Links NF-κB and Wnt/β-Catenin Pathways in Persistent Inflammatory Response Induced by Chronic Helicobacter pylori Infection

Helicobacter pylori (H. pylori) infection can cause persistent inflammatory response in human gastric mucosal epithelial cells, which may result in the occurrence of cancer. However, the underlying mechanism of carcinogenesis has not been elucidated yet. Herein, we established the models of chronic H. pylori infection in GES-1 cells and C57BL/6J mice. Interleukin 8 (IL-8) level was detected by ELISA. The expression of NF-κB p65, IL-8, Wnt2 and β-catenin mRNA and proteins was evaluated by real-time PCR, Western blotting, immunofluorescence staining, and immunohistochemistry. The infection of H. pylori in mice was evaluated by rapid urease test, H&E staining and Warthin-Starry silver staining. The morphological changes of gastric mucosa were observed by electron microscopy. Our results showed that in H. pylori infected gastric mucosal cells along with activation of NF-κB signaling pathway and increase of IL-8 level, the expression of Wnt2 was also increased significantly, which preliminarily indicates that IL-8 can positively regulate the expression of Wnt2. Studies in chronic H. pylori infected C57BL/6J mice models showed that there was an increased incidence of premalignant lesions in the gastric mucosa tissue. Through comparing changes of gastric mucosal cell ultrastructure and analyzing the relationship between NF-κB signaling pathway and Wnt2 expression, we found that H. pylori infection activated NF-κB signal pathways, and the massive release of IL-8 was positively correlated with the high expression of Wnt2 protein. Subsequently, the activated Wnt/β-catenin signal pathways may be involved in the malignant transformation of gastric mucosal cells. Collectively, H. pylori chronic infection may continuously lead to persistent inflammatory response: activate NF-κB pathway, promote IL-8 release and thereby activate Wnt/β-catenin pathway. IL-8 probably plays an important role of a linker in coupling these two signal pathways.

Orexin-A exerts neuroprotective effect in experimental intracerebral hemorrhage by suppressing autophagy via OXR1-mediated ERK/mTOR signaling pathway.

Orexin-A (OXA) is a polypeptide produced in the hypothalamus, which binds to specific receptors and exerts multiple physiological effects. Autophagy plays a vital role in early brain injury (EBI) after intracerebral hemorrhage (ICH). However, the relationship between OXA and autophagy after ICH has not been confirmed. In this study, the protective role of OXA was investigated in a model of hemin-induced injury in PC12 cells and blood-injection ICH model in rats, and its potential molecular mechanism was clarified. Neurobehavioral tests, brain water content, and pathologic morphology were assessed after ICH. Cell survival rate was determined using Cell Counting Kit-8 (CCK-8), while apoptosis was detected using flow cytometry. The autophagy protein LC3 that was originally identified as microtubule-associated protein 1 light 3 was evaluated by immunohistochemistry. The ultrastructural changes of cells following ICH were observed by transmission electron microscopy. Western blotting was performed to determine the expression levels of LC3, p62/SQSTM1 (p62), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), total extracellular signal-regulated kinase 1/2 (t-ERK1/2), mammalian target of rapamycin (mTOR), and phosphorylated mammalian target of rapamycin (p-mTOR). OXA treatment significantly improved neurofunctional outcomes, reduced brain edema, and alleviated neuronal apoptosis. OXA administration upregulated p-mTOR and p62, while it downregulated p-ERK1/2 and LC3; this effect was reversed by the orexin receptor 1 (OXR1) antagonist SB-334867. This study demonstrates that OXA suppresses autophagy via the OXR1-mediated ERK/mTOR signaling pathway to exert neuroprotective effects, and it might provide a novel therapeutic approach in patients suffering from ICH.

Toxic effects of pristine and aged polystyrene and their leachate on marine microalgae Skeletonema costatum

The acute toxic effects of pristine and aged polystyrene (P-PS and A-PS) and their leaching solutions (L-PS) on microalgae Skeletonema costatum were investigated by measuring algal density and growth inhibition rate (IR), chlorophyll concentration and photosynthetic efficiency (Fv/Fm) over 96 h. Total protein (TP), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) were measured to analyze the oxidative damage to microalgae by microplastics and their leachates. Hydrodynamic diameter of microplastics in seawater, FITR and SEM images were used to study the changes of polystyrene during aging. The interaction of algae cell with microplastics and the cellular ultrastructure changes of cells were analyzed combined with electron microscopy for a comprehensive and systematic understanding on the mechanisms of microplastic toxicity to microalgae. Both high concentration and small size of PS had significant inhibitory effect on the growth of microalgae, and the inhibitory effect was greater with increasing exposure time. The inhibition effect of aged microplastics was more obvious, which was speculated to be caused by the synergistic effect of aged PS itself and leaching solution. The negative effect of leaching solution on microalgae was due to the release of some additives during the aging process. The content of MDA reached the highest value of 54.41 nmol/mgprot in 1.0 μm 50 mg/L A-PS treatment group, and A-PS were found to be more prone to heterogeneous aggregation with algae cells by SEM.

The new preparation method for paraffin-embedded samples applying scanning electron microscopy revealed characteristic features in asthma-induced mice

In bronchial asthma patients, mucous cell metaplasia (MCM) and fibrosis occur in the bronchial epithelium and interstitium, respectively. The mucus and collagen fibers are identified by Periodic acid-Schiff stain (PAS) or Sirius red stain on optical microscopy. On a scanning electron microscope (SEM) observation, formalin-fixed-paraffin-embedded specimens have high insulation, thereby attenuating the scattered electron signals leading to insufficient contrast. Moreover, there were no staining methods for SEM observation, which characterizes the changes in epithelium and interstitium by enhancing the scattered electrons. In this study, we established a method of coating osmium thin film on pathological tissue specimens using plasma chemical vapor deposition technology. This method ensured the intensity of scattered electron signals and enabled SEM observation. Furthermore, we found that morphological changes in MCM and interstitial fibrosis could be characterized by Grocott stain, which we optimized to evaluate pathological remodeling in bronchial asthma. Using these techniques, we compared asthma-induced mice with Amphiregulin (Areg) knockout mice, and found that Areg induce MCM, but the production of Grocott-stain-positive substrate in the interstitium is Areg-independent. The method developed in this study provides an understanding of the pathological spatial information linked to the ultrastructural changes in cells and interstitium due to disease-related signaling abnormalities.

Induction of Pyroptosis in Renal Tubular Epithelial Cells Using High Glucose.

BackgroundThe micro-inflammatory state is important for the occurrence of diabetic kidney disease (DKD). Here, we aimed to explore the expression of pyroptosis related indicators and ultrastructural characteristics in DKD, and investigate pyroptosis in renal tubular epithelial cells induced by high glucose.MethodsImmunohistochemistry was used to detect expression of the inflammation-related protein NOD-like receptor protein 3 (NLRP3) and pyroptosis key protein gasdermin D (GSDMD) in kidney tissues of DKD patients. HK-2 cells were cultured in vitro and stimulated with different concentrations of glucose. The changes in HK-2 cell ultrastructure were observed using electronmicroscopy, and western blot was used to detect NLRP3, caspase-1 p20, GSDMD-N, interleukin (IL)-1β, and IL-18 expression.ResultsNLRP3 and GSDMD expression in kidney tissues of DKD patients was higher than that in control subjects. Further, GSDMD expression was positively correlated with that of NLRP3 (r = 0.847, P = 0.02). After stimulating HK-2 cells for 24 h with different glucose concentrations, compared with the control group, the 15 and 30 mmol/L glucose groups showed typical ultrastructural changes of pyroptosis. The protein expression of NLRP3, caspase-1 p20, GSDMD-N, IL-1β, and IL-18 expression in high glucose group increased significantly compared with the control group, and was glucose-concentration-dependent.ConclusionHigh glucose can activate inflammasome, cause inflammatory cytokines release, and induce pyroptosis in HK-2 cells. NLRP3-caspase-1 may be involved in GSDMD-mediated pyroptosis. This study shows a novel relationship between glucose concentration and pyroptosis, which can be studied further to design better therapies for patients with DKD.

Antibacterial efficacy and possible mechanism of action of 2-hydroxyisocaproic acid (HICA).

The exploitation of natural antimicrobial compounds that can be used in food preservation has been fast tracked by the development of antimicrobial resistance to existing antimicrobials and the increasing consumer demand for natural food preservatives. 2-hydroxyisocaproic acid (HICA) is a natural compound produced through the leucine degradation pathway and is produced in humans and by certain microorganisms such as lactic acid bacteria and Clostridium species. The present study investigated the antibacterial efficacy of HICA against some important bacteria associated with food quality and safety and provided some insights into its possible antimicrobial mechanisms against bacteria. The results revealed that HICA was effective in inhibiting the growth of tested Gram-positive and Gram-negative bacteria including a multi-drug resistant P. aeruginosa strain in this study. The underlying mechanism was investigated by measuring the cell membrane integrity, membrane permeability, membrane depolarisation, and morphological and ultrastructural changes after HICA treatment in bacterial cells. The evidence supports that HICA exerts its activity via penetration of the bacterial cell membranes, thereby causing depolarisation, rupture of membranes, subsequent leakage of cellular contents and cell death. The current study suggests that HICA has potential to be used as an antibacterial agent against food spoilage and food-borne pathogenic bacteria, targeting the bacterial cell envelope.

P45 Antimicrobial activity of ruthenium-based metallo-therapeutics against Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is highly resistant to antibiotics and biocidal products used in both medical and industrial environments respectively. Metal-based compounds have been used as antimicrobial agents throughout history for a broad range of applications. More recently, it has been shown that ruthenium (Ru)-based compounds have potent antimicrobial properties and, in contrast to traditional antibiotics, these are thought to elicit antibacterial activity at multiple sites within the bacterial cell, which will undoubtedly reduce the possibility of resistance evolution.MethodsMIC and MBC assays coupled with disc diffusion assays were used to screen a library of Ru-based compounds.ResultsOne lead compound was identified that was highly active at inhibiting growth of multiple strains of P. aeruginosa at ≤32 mg/L. Crystal violet biofilm assays were performed, which showed a decrease in biomass following exposure over a 24 h period. Scanning electron microscopy was used to reveal morphological changes in the bacterial cell ultrastructure when exposed to the Ru-based compound, with evidence of membrane perturbation that supported a proposed mechanism of antimicrobial activity.ConclusionsThese findings make a significant contribution towards the search for novel bactericidal agents and further research is now focused on determining the potential for use as novel adjuvants within medicinal applications such as wound care management.

Transmission Electron Microscopy to Follow Ultrastructural Modifications of Erythroblasts Upon ex vivo Human Erythropoiesis

Throughout mammal erythroid differentiation, erythroblasts undergo enucleation and organelle clearance becoming mature red blood cell. Organelles are cleared by autophagic pathways non-specifically targeting organelles and cytosolic content or by specific mitophagy targeting mitochondria. Mitochondrial functions are essential to coordinate metabolism reprogramming, cell death, and differentiation balance, and also synthesis of heme, the prosthetic group needed in hemoglobin assembly. In mammals, mitochondria subcellular localization and mitochondria interaction with other structures as endoplasmic reticulum and nucleus might be of importance for the removal of the nucleus, that is, the enucleation. Here, we aim to characterize by electron microscopy the changes in ultrastructure of cells over successive stages of human erythroblast differentiation. We focus on mitochondria to gain insights into intracellular localization, ultrastructure, and contact with other organelles. We found that mitochondria are progressively cleared with a significant switch between PolyE and OrthoE stages, acquiring a rounded shape and losing contact sites with both ER (MAM) and nucleus (NAM). We studied intracellular vesicle trafficking and found that endosomes and MVBs, known to be involved in iron traffic and heme synthesis, are increased during BasoE to PolyE transition; autophagic structures such as autophagosomes increase from ProE to OrthoE stages. Finally, consistent with metabolic switch, glycogen accumulation was observed in OrthoE stage.